Protocol for isolating CD163+ Kupffer cells from human liver resections.

The liver microenvironment contains a wide variety of monocyte and macrophage populations. Here, we present a protocol for the specific isolation of liver-resident macrophages, known as Kupffer cells (KCs), from human liver resections. We describe steps for dissociating human liver tissues, separating non-parenchymal cells into fractions by a 2-phase iodixanol gradient, and positive selection of KCs based on the expression of CD163. We then provide instructions for validating the procedure by immunofluorescence to detect CD163. For complete details on the use and execution of this protocol, please refer to Roca Suarez, Plissonnier, et al.1.

Applications of CRISPR/Cas as a Toolbox for Hepatitis B Virus Detection and Therapeutics.

Hepatitis B virus (HBV) infection remains a significant global health challenge, leading to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Covalently closed circular DNA (cccDNA) and integrated HBV DNA are pivotal in maintaining viral persistence. Recent advances in CRISPR/Cas technology offer innovative strategies to inhibit HBV by directly targeting both cccDNA and integrated HBV DNA or indirectly by degrading HBV RNAs or targeting host proteins. This review provides a comprehensive overview of the latest advancements in using CRISPR/Cas to inhibit HBV, with a special highlight on newer non-double-strand (non-DSB) break approaches. Beyond the canonical use of CRISPR/Cas for target inhibition, we discuss additional applications, including HBV diagnosis and developing models to understand cccDNA biology, highlighting the diverse use of this technology in the HBV field.

A molecular standard for circulating HBV RNA detection and quantification assays in patients with chronic hepatitis B.

Background & Aims: Circulating HBV RNAs have been proposed as a biomarker that reflects the transcriptional activity of covalently closed circular DNA (cccDNA) and may help to evaluate HBV treatment activity. Different research assays have been proposed and, although two PCR-based research use only investigational assays have been developed, the lack of standardized protocols represents an important limitation. Here we have designed and generated a stable clonal cell line producing an RNA-based standard for the calibration of PCR-based circulating HBV RNA assays. Methods: HBV RNA-producing Huh7-derived stable cell lines were generated by transfecting pTriEX plasmids containing 1.1 unit length HBV DNA genomes carrying mutations in the catalytic site (YMAA mutation) and the TP domain (Y63F) of the polymerase, and the ε-loop of the pregenomic (pg)RNA (mutation A1G). Results: The clonal cell line (Huh7-3D29), carrying a double YMAA and Y63F mutation, displayed, and maintained over several passages in culture, a high RNA secretion phenotype with negligible residual secreted HBV DNA. Density gradient centrifugation showed that most of the secreted HBV RNA from Huh7-3D29 cells was detected in naked capsid and virion-like particles and only a minority in small extracellular vescicles. Nanopore sequencing of 5'RACE products shows that the majority of the Huh7-3D29-secreted HBV RNAs start at the 5' end of pgRNA and pgRNA-derived spliced RNAs. Finally, Huh7-3D29 cells showed a high and up-scalable secreted RNA yield allowing 1,300 standard curves in 9 days from one flask. Conclusion: We generated a clonal cell line that produces high quantities of HBV RNAs with very low quantities of contaminating HBV DNAs, representing a stable source of RNA standard for HBV RNA assay calibration. Impact and implications: Several investigational assays and two research use only assays have been developed to detect and quantify circulating HBV RNAs, an emerging biomarker of covalently closed circular DNA transcriptional activity and target engagement by new HBV treatments. The lack of a unique molecular standard for circulating HBV RNA quantification represents an important limitation. Here we describe the generation of a stable clonal cell line producing and secreting an RNA-based standard containing all the HBV RNA species found in HBV patients' sera (e.g. pgRNA, HBx transcripts). This new RNA standard can be used to calibrate all PCR-based assays for circulating HBV RNA quantification to evaluate, in a non-invasive manner, the size of the transcriptionally active cccDNA pool and the activity of novel strategies aimed at curing HBV infection.

After the Storm: Persistent Molecular Alterations Following HCV Cure.

The development of direct-acting antivirals (DAAs) against hepatitis C virus (HCV) has revolutionized the management of this pathology, as their use allows viral elimination in a large majority of patients. Nonetheless, HCV remains a major public health problem due to the multiple challenges associated with its diagnosis, treatment availability and development of a prophylactic vaccine. Moreover, HCV-cured patients still present an increased risk of developing hepatic complications such as hepatocellular carcinoma. In the present review, we aim to summarize the impact that HCV infection has on a wide variety of peripheral and intrahepatic cell populations, the alterations that remain following DAA treatment and the potential molecular mechanisms implicated in their long-term persistence. Finally, we consider how recent developments in single-cell multiomics could refine our understanding of this disease in each specific intrahepatic cell population and drive the field to explore new directions for the development of chemo-preventive strategies.

Detection and Quantification of HBV Transcripts by Full-Length 5'RACE-PCR.

Chronic hepatitis B virus infection is a major health burden worldwide. Although efficient in maintaining the infection under control, current treatments are unable to fully eradicate the virus due to the persistence of its minichromosome, the so-called cccDNA. In the context of emerging antiviral and combinatorial therapies aiming at decreasing the cccDNA pool or at silencing its transcriptional activity, the detection and quantification of viral RNAs have gained increasing interest as a way to monitor the HBV reservoir. Here, we describe the protocol of the HBV full-length 5'RACE, a technique that allows to define the repertoire of the different HBV RNA species in vitro, intracellularly and extracellularly, and in vivo, in patients' serum in a qualitative or quantitative manner, depending on the choice of the post-analysis methodology.

Helicases DDX5 and DDX17 promote heterogeneity in HBV transcription termination in infected human hepatocytes.

BACKGROUND & AIMS: Transcription termination fine-tunes gene expression and contributes to the specification of RNA function in eukaryotic cells. Transcription termination of HBV is subject to the recognition of the canonical polyadenylation signal (cPAS) common to all viral transcripts. However, the regulation of this cPAS and its impact on viral gene expression and replication is currently unknown. METHODS: To unravel the regulation of HBV transcript termination, we implemented a 3' RACE (rapid amplification of cDNA ends)-PCR assay coupled to single molecule sequencing both in in vitro-infected hepatocytes and in chronically infected patients. RESULTS: The detection of a previously unidentified transcriptional readthrough indicated that the cPAS was not systematically recognized during HBV replication in vitro and in vivo. Gene expression downregulation experiments demonstrated a role for the RNA helicases DDX5 and DDX17 in promoting viral transcriptional readthrough, which was, in turn, associated with HBV RNA destabilization and decreased HBx protein expression. RNA and chromatin immunoprecipitation, together with mutation of the cPAS sequence, suggested a direct role of DDX5 and DDX17 in functionally linking cPAS recognition to transcriptional readthrough, HBV RNA stability and replication. CONCLUSIONS: Our findings identify DDX5 and DDX17 as crucial determinants of HBV transcriptional fidelity and as host restriction factors for HBV replication. IMPACT AND IMPLICATIONS: HBV covalently closed circular (ccc)DNA degradation or functional inactivation remains the holy grail for the achievement of HBV cure. Transcriptional fidelity is a cornerstone in the regulation of gene expression. Here, we demonstrate that two helicases, DDX5 and DDX17, inhibit recognition of the HBV polyadenylation signal and thereby transcriptional termination, thus decreasing HBV RNA stability and acting as restriction factors for efficient cccDNA transcription and viral replication. The observation that DDX5 and DDX17 are downregulated in patients chronically infected with HBV suggests a role for these helicases in HBV persistence in vivo. These results open new perspectives for researchers aiming at identifying new targets to neutralise cccDNA transcription.

TLR8 agonist selgantolimod regulates Kupffer cell differentiation status and impairs HBV entry into hepatocytes via an IL-6-dependent mechanism.

OBJECTIVE: Achieving HBV cure will require novel combination therapies of direct-acting antivirals and immunomodulatory agents. In this context, the toll-like receptor 8 (TLR8) agonist selgantolimod (SLGN) has been investigated in preclinical models and clinical trials for chronic hepatitis B (CHB). However, little is known regarding its action on immune effectors within the liver. Our aim was to characterise the transcriptomic changes and intercellular communication events induced by SLGN in the hepatic microenvironment. DESIGN: We identified TLR8-expressing cell types in the human liver using publicly available single-cell RNA-seq data and established a method to isolate Kupffer cells (KCs). We characterised transcriptomic and cytokine KC profiles in response to SLGN. SLGN's indirect effect was evaluated by RNA-seq in hepatocytes treated with SLGN-conditioned media (CM) and quantification of HBV parameters following infection. Pathways mediating SLGN's effect were validated using transcriptomic data from HBV-infected patients. RESULTS: Hepatic TLR8 expression takes place in the myeloid compartment. SLGN treatment of KCs upregulated monocyte markers (eg, S100A12) and downregulated genes associated with the KC identity (eg, SPIC). Treatment of hepatocytes with SLGN-CM downregulated NTCP and impaired HBV entry. Cotreatment with an interleukin 6-neutralising antibody reverted the HBV entry inhibition. CONCLUSION: Our transcriptomic characterisation of SLGN sheds light into the programmes regulating KC activation. Furthermore, in addition to its previously described effect on established HBV infection and adaptive immunity, we show that SLGN impairs HBV entry. Altogether, SLGN may contribute through KCs to remodelling the intrahepatic immune microenvironment and may thus represent an important component of future combinations to cure HBV infection.

Circulating HBV RNA and Hepatitis B Core-Related Antigen Trajectories in Persons With HIV/HBV Coinfection and Hepatitis B Surface Antigen Loss During Tenofovir Therapy.

BACKGROUND: We evaluated long-term trajectories of circulating hepatitis B virus (HBV) RNA and hepatitis B core-related antigen (HBcrAg) in persons with and without hepatitis B surface antigen (HBsAg) loss during tenofovir therapy in the Swiss HIV Cohort Study. METHODS: We included 29 persons with HIV with HBsAg loss and 29 matched persons with HIV without HBsAg loss. We compared HBV RNA and HBcrAg decline and assessed the cumulative proportions with undetectable HBV RNA and HBcrAg levels during tenofovir therapy using Kaplan-Meier estimates. RESULTS: HBsAg loss occurred after a median of 4 years (IQR, 1-8). All participants with HBsAg loss achieved suppressed HBV DNA and undetectable HBV RNA preceding undetectable quantitative HBsAg levels, whereas 79% achieved negative HBcrAg. In comparison, 79% of participants without HBsAg loss achieved undetectable HBV-RNA and 48% negative HBcrAg. After 2 years of tenofovir therapy, an HBV RNA decline ≥1 log10 copies/mL had 100% sensitivity and 36.4% specificity for HBsAg loss, whereas an HBcrAg decline ≥1 log10 U/mL had 91.0% sensitivity and 64.5% specificity. CONCLUSIONS: HBV RNA suppression preceded undetectable quantitative HBsAg levels and had high sensitivity but low specificity for HBsAg loss during tenofovir therapy in persons with HIV. HBcrAg remained detectable in approximately 20% of persons with HBsAg loss and 50% of persons without HBsAg loss.

Co-Transcriptional Regulation of HBV Replication: RNA Quality Also Matters.

Chronic hepatitis B (CHB) virus infection is a major public health burden and the leading cause of hepatocellular carcinoma. Despite the efficacy of current treatments, hepatitis B virus (HBV) cannot be fully eradicated due to the persistence of its minichromosome, or covalently closed circular DNA (cccDNA). The HBV community is investing large human and financial resources to develop new therapeutic strategies that either silence or ideally degrade cccDNA, to cure HBV completely or functionally. cccDNA transcription is considered to be the key step for HBV replication. Transcription not only influences the levels of viral RNA produced, but also directly impacts their quality, generating multiple variants. Growing evidence advocates for the role of the co-transcriptional regulation of HBV RNAs during CHB and viral replication, paving the way for the development of novel therapies targeting these processes. This review focuses on the mechanisms controlling the different co-transcriptional processes that HBV RNAs undergo, and their contribution to both viral replication and HBV-induced liver pathogenesis.

Major open questions in the hepatitis B and D field - Proceedings of the inaugural International emerging hepatitis B and hepatitis D researchers workshop.

The early and mid-career researchers (EMCRs) of scientific communities represent the forefront of research and the future direction in which a field takes. The opinions of this key demographic are not commonly aggregated to audit fields and precisely demonstrate where challenges lie for the future. To address this, we initiated the inaugural International Emerging Researchers Workshop for the global Hepatitis B and Hepatitis D scientific community (75 individuals). The cohort was split into small discussion groups and the significant problems, challenges, and future directions were assessed. Here, we summarise the outcome of these discussions and outline the future directions suggested by the EMCR community. We show an effective approach to gauging and accumulating the ideas of EMCRs and provide a succinct summary of the significant gaps remaining in the Hepatitis B and Hepatitis D field.

Cytosine base editing inhibits hepatitis B virus replication and reduces HBsAg expression in vitro and in vivo.

Chronic hepatitis B virus (HBV) infection remains a global health problem due to the lack of treatments that prevent viral rebound from HBV covalently closed circular (ccc)DNA. In addition, HBV DNA integrates in the human genome, serving as a source of hepatitis B surface antigen (HBsAg) expression, which impairs anti-HBV immune responses. Cytosine base editors (CBEs) enable precise conversion of a cytosine into a thymine within DNA. In this study, CBEs were used to introduce stop codons in HBV genes, HBs and Precore. Transfection with mRNA encoding a CBE and a combination of two guide RNAs led to robust cccDNA editing and sustained reduction of the viral markers in HBV-infected HepG2-NTCP cells and primary human hepatocytes. Furthermore, base editing efficiently reduced HBsAg expression from HBV sequences integrated within the genome of the PLC/PRF/5 and HepG2.2.15 cell lines. Finally, in the HBV minicircle mouse model, using lipid nanoparticulate delivery, we demonstrated antiviral efficacy of the base editing approach with a >3log10 reduction in serum HBV DNA and >2log10 reduction in HBsAg, and 4/5 mice showing HBsAg loss. Combined, these data indicate that base editing can introduce mutations in both cccDNA and integrated HBV DNA, abrogating HBV replication and silencing viral protein expression.

Quantification of circulating HBV RNA expressed from intrahepatic cccDNA in untreated and NUC treated patients with chronic hepatitis B.

OBJECTIVE: A convenient, reproducible biomarker of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) transcriptional activity is lacking. We measured circulating HBV RNA (cirB-RNA) in untreated and nucleos(t)ide analogues (NUC) treated chronic hepatitis B (CHB) patients to define its correlation with intrahepatic viral markers and HBV core-related antigen (HBcrAg). DESIGN: Paired liver biopsy and serum samples were collected from 122 untreated and 30 NUC-treated CHB patients. We measured cirB-RNA, HBV DNA, hepatitis B surface antigen (HBsAg), HBcrAg and alanine aminotransferase levels. cirB-RNA was quantified using an investigational HBV RNA assay for use on the cobas 6800 system. The test detects a region spanning the HBV canonical polyadenylation site. cccDNA and 3.5 kb RNA in liver tissue were assessed by quantitative PCR and droplet digital PCR. RESULTS: cirB-RNA was detectable in 100% of HBeAg(+) chronic hepatitis (CH), 57% and 14% of HBeAg(-) CH and chronic infection untreated patients and 47% of NUC-treated patients. cirB-RNA undetectability was associated with lower intrahepatic cccDNA transcriptional activity, as well as serum HBcrAg, but no significant differences in HBsAg, in both untreated and treated patients. In untreated HBeAg(-) patients, cirB-RNA correlated with intrahepatic 3.5 kb RNA and cccDNA transcriptional activity, serum HBV DNA and HBcrAg, but not with HBsAg or total cccDNA levels. Combined undetectability of both cirB-RNA and HBcrAg detection in untreated HBeAg(-) patients identified a subgroup with the lowest levels of intrahepatic transcriptionally active cccDNA. CONCLUSION: Our results support the usefulness of quantification of circulating HBV RNA expressed from cccDNA as an indicator of intrahepatic active viral reservoir in both untreated and NUC-treated CHB patients. TRIAL REGISTRATION NUMBER: NCT02602847.

G-quadruplexes control hepatitis B virus replication by promoting cccDNA transcription and phase separation in hepatocytes.

Phase separation regulates fundamental processes in gene expression and is mediated by the local concentration of proteins and nucleic acids, as well as nucleic acid secondary structures such as G-quadruplexes (G4s). These structures play fundamental roles in both host gene expression and in viral replication due to their peculiar localisation in regulatory sequences. Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is an episomal minichromosome whose persistence is at the basis of chronic infection. Identifying the mechanisms controlling its transcriptional activity is indispensable to develop new therapeutic strategies against chronic hepatitis B. The aim of this study was to determine whether G4s are formed in cccDNA and regulate viral replication. Combining biochemistry and functional studies, we demonstrate that cccDNA indeed contains ten G4s structures. Furthermore, mutations disrupting two G4s located in the enhancer I HBV regulatory region altered cccDNA transcription and viral replication. Finally, we showed for the first time that cccDNA undergoes phase separation in a G4-dependent manner to promote its transcription in infected hepatocytes. Altogether, our data give new insight in the transcriptional regulation of the HBV minichromosome that might pave the way for the identification of novel targets to destabilize or silence cccDNA.

Evaluation of the HBV liver reservoir with fine needle aspirates.

Background & Aims: Finite duration of treatment associated with HBsAg loss is the current goal for improved therapeutic approaches against chronic HBV infection, as it indicates elimination or durable inactivation of intrahepatic covalently closed circular DNA (cccDNA). To assist drug development, the definition of early predictive markers of HBsAg loss by assessing their value in reflecting intrahepatic cccDNA levels and transcriptional activity is essential. Fine needle aspirates (FNAs) have recently emerged as a less invasive alternative to core liver biopsy (CLB) and showed to be useful for investigating intrahepatic immune responses. The aim of this study was to optimise and validate the use of FNA vs. CLB to evaluate the intrahepatic viral reservoir. Methods: Paired FNA/CLB samples were obtained from patients with HBeAg+ chronic hepatitis (n = 4), HBeAg- chronic hepatitis (n = 4), and HBeAg- chronic infection (n = 1). One HBeAg+ patient was undergoing tenofovir treatment. HBV 3.5-kb RNA and cccDNA were quantified by droplet digital PCR. Results: cccDNA was quantifiable in all but one FNA/CLB pair, showing the highest levels in untreated HBeAg+ patients, except for the tenofovir-treated patient. Similarly, 3.5-kb RNA was detectable in all but one FNA sample and showed higher levels in HBeAg+ patients. When comparing cccDNA and 3.5-kb RNA quantification in FNA vs. CLB samples, no statistically significant differences were identified. Conclusions: These results demonstrate the possibility to quantify cccDNA and assess its transcriptional activity in patients with chronic hepatitis B by combining FNA and droplet digital PCR. This supports the use of FNA in clinical trials to evaluate the intrahepatic viral reservoir during the development of new antivirals and immunomodulatory agents. Impact and implications: Chronic hepatitis B infection is characterised by a complex interplay between immune responses and viral replication in the liver, which determines the long-term outcome of the disease. In this study, we show that fine needle aspiration of the liver, a less-invasive alternative to core biopsies, allows the assessment of the hepatic viral reservoir.

Interspecies comparison of the early transcriptomic changes associated with hepatitis B virus exposure in human and macaque immune cell populations.

Background and aims: Hepatitis B virus (HBV) infection affects 300 million individuals worldwide, representing a major factor for the development of hepatic complications. Although existing antivirals are effective in suppressing replication, eradication of HBV is not achieved. Therefore, a multi-faceted approach involving antivirals and immunomodulatory agents is required. Non-human primates are widely used in pre-clinical studies due to their close evolutionary relationship to humans. Nonetheless, it is fundamental to identify the differences in immune response between humans and these models. Thus, we performed a transcriptomic characterization and interspecies comparison of the early immune responses to HBV in human and cynomolgus macaques. Methods: We characterized early transcriptomic changes in human and cynomolgus B cells, T cells, myeloid and plasmacytoid dendritic cells (pDC) exposed to HBV ex vivo for 2 hours. Differentially-expressed genes were further compared to the profiles of HBV-infected patients using publicly-available single-cell data. Results: HBV induced a wide variety of transcriptional changes in all cell types, with common genes between species representing only a small proportion. In particular, interferon gamma signaling was repressed in human pDCs. At the gene level, interferon gamma inducible protein 16 (IFI16) was upregulated in macaque pDCs, while downregulated in humans. Moreover, IFI16 expression in pDCs from chronic HBV-infected patients anti-paralleled serum HBsAg levels. Conclusion: Our characterization of early transcriptomic changes induced by HBV in humans and cynomolgus macaques represents a useful resource for the identification of shared and divergent host responses, as well as potential immune targets against HBV.

Early intrahepatic recurrence of HBV infection in liver transplant recipients despite antiviral prophylaxis.

Background & Aims: Prophylaxis with nucleos(t)ide analogues (NUCs) and hepatitis B immunoglobulin (HBIG) has decreased the rate of HBV recurrence after orthotopic liver transplantation (OLT), but the duration of this prophylaxis remains debated. Our aim was to investigate the recurrence of both intrahepatic and serum HBV markers after OLT in patients receiving long-term NUC and HBIG prophylaxis. Methods: A total of 31 HBV-positive patients benefiting from OLT were prospectively enrolled in five French centres between 2012 and 2015. Tissue samples from the native liver, liver reperfusion biopsy, and 12-month post-OLT (M12) biopsy were collected. Intrahepatic HBV markers were quantified using Droplet Digital PCR. Serum hepatitis B core-related antigen (HBcrAg) and HBsAg were quantified using the Lumipulse platform. Results: Among the 31 patients, 26 were HBeAg negative and 28 had undetectable serum HBV DNA at OLT. All patients received HBIG and NUC after OLT, and serum HBV DNA was undetectable at M12. Of the 27 available native livers, 26 had detectable total HBV DNA (median, 0.045 copies/cell), 21 were positive for cccDNA (0.001 copies/cell), and 19 were positive for 3.5-kb HBV RNA (0.0004 copies/cell). Among the 14 sequential reperfusion and M12 biopsies, seven were positive for HBV markers on the reperfusion sampling, and six of them were also positive at M12. Of the 27 patients with available serum samples at M12, eight were positive for HBcrAg and five were positive for HBsAg by ultrasensitive quantification, although they were negative by conventional techniques. Overall, among the 17 patients having a matched biopsy and serum sample at M12, only one had undetectable HBV markers in both the liver and serum. Conclusions: Our results demonstrate a very early detection of viral genome in the graft and intrahepatic viral recurrence despite NUC and HBIG prophylaxis. Clinical Trials Registration: This study is registered at ClinicalTrials.gov (NCT02602847). Impact and Implications: In this work, we show that, despite the recommended prophylaxis based on NUC and HBIG, HBV can infect the new liver very rapidly after transplantation. Twelve months after transplantation, the majority of patients had at least one HBV marker detected in either serum or the liver. Therefore, our results demonstrate early intrahepatic viral recurrence despite NUC and HBIG therapy and underline the importance of an optimal patient compliance to the antiviral prophylaxis to prevent viral rebound.

Emerging anti-HDV drugs and HBV cure strategies with anti-HDV activity.

Hepatitis delta virus (HDV) is a satellite RNA virus that requires the presence of hepatitis B virus (HBV) for its replication. HDV/HBV co-infection is often associated with a faster disease progression of chronic hepatitis in comparison to HBV mono-infection. Therefore, the development of novel antiviral therapies targeting HDV represents a high priority and an urgent medical need. In this review, we summarize the ongoing efforts to evaluate promising HDV-specific drugs, such as lonafarnib (LNF), pegylated interferon lambda (PEG-IFN-λ) and their use as a combination therapy. Furthermore, we review the most recent developments in the area of anti-HBV drugs with potential effects against HDV, including therapeutic agents targeting hepatitis B surface antigen (HBsAg) expression, secretion and function. Finally, we consider the important insights that have emerged from the development of these potential antiviral strategies, as well as the intriguing questions that remain to be elucidated in this rapidly changing field.

Hepatitis B Virus Targets Lipid Transport Pathways to Infect Hepatocytes.

BACKGROUND & AIMS: A single hepatitis B virus (HBV) particle is sufficient to establish chronic infection of the liver after intravenous injection, suggesting that the virus targets hepatocytes via a highly efficient transport pathway. We therefore investigated whether HBV uses a physiological liver-directed pathway that supports specific host-cell targeting in vivo. METHODS: We established the ex vivo perfusion of intact human liver tissue that recapitulates the liver physiology to investigate HBV liver targeting. This model allowed us to investigate virus-host cell interactions in a cellular microenvironment mimicking the in vivo situation. RESULTS: HBV was rapidly sequestered by liver macrophages within 1 hour after a virus pulse perfusion but was detected in hepatocytes only after 16 hours. We found that HBV associates with lipoproteins in serum and within machrophages. Electron and immunofluorescence microscopy corroborated a co-localization in recycling endosomes within peripheral and liver macrophages. Recycling endosomes accumulated HBV and cholesterol, followed by transport of HBV back to the cell surface along the cholesterol efflux pathway. To reach hepatocytes as final target cells, HBV was able to utilize the hepatocyte-directed cholesterol transport machinery of macrophages. CONCLUSIONS: Our results propose that by binding to liver targeted lipoproteins and using the reverse cholesterol transport pathway of macrophages, HBV hijacks the physiological lipid transport pathways to the liver to most efficiently reach its target organ. This may involve transinfection of liver macrophages and result in deposition of HBV in the perisinusoidal space from where HBV can bind its receptor on hepatocytes.