Analysis of a cellular model to account for the natural history of infection by the hepatitis B virus and its role in the development of primary hepatocellular carcinoma.

Infection with the hepatitis B virus (HBV) can have many different outcomes. Transient infection may result in acute hepatitis or may remain subclinical. Persistent infection may also be subclinical, or may involve chronic active hepatitis, and can finally lead to the development of primary hepatocellular carcinoma. A mathematical model is given to account for the many different outcomes of HBV pathogenesis. The model is based on the assumption that the liver contains two cell populations with differing abilities to support active HBV replication and/or viral integration into the genome. The model helps account for the relationship of the different clinical courses of HBV infection to the age when the disease is acquired, together with the state of the immune system of the patient.     

乙型肝炎病毒与细胞相互作用的研究进展

乙型肝炎病毒( HBV) 是一种类反转录( retroid) 病毒, 其DNA 基因组短小, 编码能力有限。但HBV 却能成功地在不同宿主个体中繁衍, 并在人群中广泛传播。本文着重对HBV 与宿主细胞关系的研究进展进行综述, 以利于阐明HBV 复制及其在宿主细胞内持续存在的特性。这些内容主要来自于本实验室的研究。文中讨论的HBV 与宿主之间的相互作用主要包括: 细胞伴侣分子在HBV 反转录起始和核衣壳组装中的作用,宿主通过调控核衣壳磷酸化对病毒衣壳成熟的调节以及与HBV 持续存在相关的核内附加体( episomal) 病毒DNA 的形成。     

Control or persistence of hepatitis B virus: the critical role of initial host-virus interactions

Following infection with hepatitis B virus (HBV), the period before symptomatic disease is now recognized as a time of dynamic interaction between virus and host. Recent work has shown that this period is the phase of infection during which maximal changes in virus replication and the activation of critical components of the immune system occurs. This suggests that the different outcomes following exposure might be determined during the early phase of infection, before the onset of clinical disease. The hypothesis that small differences in the dynamic relationship between host and virus, such as in the kinetics of HBV replication, may influence the final outcome of infection, will be discussed.     

Modelling Cooperation between fishermen with a Cellular automaton. A framework for fishing effort spatial dynamics

In many fisheries sharing information between vessels is an important characteristic of fishermen's behaviour rarely modelled or analyzed. A Cellular Automaton is designed in an attempt to understand circumstances that favour group formation. The simulated world is toroidal with a static fishing resource distributed in patches. Movement decisions are random in the case of fishermen in a local scale. After a certain time interval, sharing information is possible between fishermen in a dynamic Moore's neighbourhood of cells at a broader scale and movement to adjacent areas may occur according to a set of rules. The dynamic neighbourhood is a novel concept defined in this work within the framework of Cellular Automata. Decision making by each fisherman is a function of the influence other fishermen (neighbours) exert on them as well as on personal knowledge, to form an opinion of the areas (cells) quality, and take action consequently.     

Feedback Circuits in Hepatitis B Virus Infection

A simplified model using kinetic logic is proposed to approach the problem after Hepatitis B viral (HBV) infection. It accounts for several stable regimes or attractors corresponding to the essential dynamic behaviour of the replication of the Hepatitis B virus. Infection with the virus can result in viral clearance, fulminant hepatic failure and death, or chronic transmissible infection, that is multistationarity corresponding to the existence of the positive feedback circuit in our modelling. Another implication of this model is the existence of oscillations or homeostatic mechanisms, sometimes observed in the viral cycle, consistent with the existence of the negative feedback circuit. Thus, this report shows how a simple model of kinetic logic may be used to account for the variety of manifestations of HBV infection. This model implies the presence of the Hepatitis B e antigen, whose conservation suggests that it plays an important role in the life cycle of hepadnaviruses. Its function in the viral cycle is still unknown, but our model suggests that this antigen could explain the passage from one state of the viral infection (acute or latent) to another, as well as the oscillatory behavior which may account for the intermittent symptoms of hepatitis observed in some patients. Furthermore, this model shows a virgin state. This state is also reached after recovery. The model proposed demonstrates that starting from a viral acute infection, the host's immune response, depending on the immunological status of the patient, can lead to viral clearance, or to periodic spontaneous reactivation.     

Inhibitory effect of silibinin on hepatitis B virus entry

Hepatitis B virus (HBV) infection is a worldwide health problem because of its potential to cause liver cirrhosis and hepatocellular carcinoma. Silibinin is a constituent of an extract of milk thistle, which is empirically used as a herbal medicine for the protection of liver, but its detailed effects on HBV are unknown. Because a previous study reported that silibinin hinders clathlin-mediated endocytosis (CME), we aimed to test whether silibinin inhibits the entry of HBV into hepatocytes. Using HepG2-NTCP-C4 cells, which overexpress sodium taurocholate cotransporting polypeptide (NTCP), it was shown that silibinin inhibited HBV infection dose-dependently. Similar effects were observed using human primary hepatocytes (PXB-cells). Additionally, a combination of silibinin and entecavir reduced HBV DNA in the culture supernatant more than either mono-treatment alone in HepG2-NTCP-C4 cells that had already been infected with HBV. Silibinin decreased transferrin uptake but did not affect the interaction between the HBV envelope and NTCP, suggesting that silibinin might inhibit HBV infection by hindering CME. In conclusion, this study showed that silibinin inhibits HBV entry in vitro.     

Influence of backward bifurcation in a model of hepatitis B and C viruses

In the 1990s, liver transplantation for hepatitis B and C virus (HBV and HCV) related-liver diseases was a very controversial issue since recurrent infection of the graft was inevitable. Significant progress has been made in the prophylaxis and treatment of recurrent hepatitis B/C (or HBV/HCV infection) after liver transplantation. In this paper, we propose a mathematical model of ordinary differential equations describing the dynamics of the HBV/HCV and its interaction with both liver and blood cells. A single model is used to describe infection of either virus since the dynamics in-host (infected of the liver) are similar. Analyzing the model, we observe that the system shows either a transcritical or a backward bifurcation. Explicit conditions on the model parameters are given for the backward bifurcation to be present. Consequently, we investigate possible factors that are responsible for HBV/HCV infection and assess control strategies to reduce HBV/HCV reinfection and improve graft survival after liver transplantation.     

Turnover of hepatitis B virus X protein is facilitated by Hdj1, a human Hsp40/DnaJ protein

Hepatitis B virus X (HBX) protein is required for the productive infection of hepatitis B virus (HBV) in vivo and implicated in the development of hepatocellular carcinoma. We have previously shown that hTid-1 and Hdj1, the human Hsp40/DnaJ chaperone proteins, bind the HBV core protein and inhibit viral replication in cell culture system. Here, we report evidences to suggest that HBX is the major target of Hdj1 in the inhibition of HBV replication. Expression of Hdj1 in cultured human hepatoma HepG2 cells facilitated degradation of HBX by the proteasome pathway, and thereby inhibited replication of the wild-type HBV as well as that of the HBX-deficient mutant virus rescued by HBX supplied in trans. Mutational analyses indicated that J domain of Hdj1 is required for the process. These results might provide a molecular basis for the antiviral effect of cellular chaperones.     

Viral hepatitis markers in antepartum and postpartum women in Rio de Janeiro,Brazil

A seroprevalence study was carried out among a group of women in Rio de Janeiro to determine the prevalence of different markers for viral hepatitis given the limited data among healthy populations. Blood samples collected and tested from 874 women before or after delivery in a public county maternity hospital demonstrated age to be directly related to markers for hepatitis A virus and hepatitis B virus (HBV) infection. The prevalence of HBV and hepatitis C virus infection were lower than that observed in the blood donor population and might be explained by the younger age group and gender.     

Proteoglycans Act as Cellular Hepatitis Delta Virus Attachment Receptors

The hepatitis delta virus (HDV) is a small, defective RNA virus that requires the presence of the hepatitis B virus (HBV) for its life cycle. Worldwide more than 15 million people are co-infected with HBV and HDV. Although much effort has been made, the early steps of the HBV/HDV entry process, including hepatocyte attachment and receptor interaction are still not fully understood. Numerous possible cellular HBV/HDV binding partners have been described over the last years; however, so far only heparan sulfate proteoglycans have been functionally confirmed as cell-associated HBV attachment factors. Recently, it has been suggested that ionotrophic purinergic receptors (P2XR) participate as receptors in HBV/HDV entry. Using the HBV/HDV susceptible HepaRG cell line and primary human hepatocytes (PHH), we here demonstrate that HDV entry into hepatocytes depends on the interaction with the glycosaminoglycan (GAG) side chains of cellular heparan sulfate proteoglycans. We furthermore provide evidence that P2XR are not involved in HBV/HDV entry and that effects observed with inhibitors for these receptors are a consequence of their negative charge. HDV infection was abrogated by soluble GAGs and other highly sulfated compounds. Enzymatic removal of defined carbohydrate structures from the cell surface using heparinase III or the obstruction of GAG synthesis by sodium chlorate inhibited HDV infection of HepaRG cells. Highly sulfated P2XR antagonists blocked HBV/HDV infection of HepaRG cells and PHH. In contrast, no effect on HBV/HDV infection was found when uncharged P2XR antagonists or agonists were applied. In summary, HDV infection, comparable to HBV infection, requires binding to the carbohydrate side chains of hepatocyte-associated heparan sulfate proteoglycans as attachment receptors, while P2XR are not actively involved.     

Spinoculation Enhances HBV Infection in NTCP-Reconstituted Hepatocytes

Hepatitis B virus (HBV) infection and its sequelae remain a major public health burden, but both HBV basic research and the development of antiviral therapeutics have been hindered by the lack of an efficient in vitro infection system. Recently, sodium taurocholate cotransporting polypeptide (NTCP) has been identified as the HBV receptor. We herein report that we established a NTCP-complemented HepG2 cell line (HepG2-NTCP12) that supports HBV infection, albeit at a low infectivity level following the reported infection procedures. In our attempts to optimize the infection conditions, we found that the centrifugation of HepG2-NTCP12 cells during HBV inoculation (termed “spinoculation”) significantly enhanced the virus infectivity. Moreover, the infection level gradually increased with accelerated speed of spinoculation up to 1,000g tested. However, the enhancement of HBV infection was not significantly dependent upon the duration of centrifugation. Furthermore, covalently closed circular (ccc) DNA was detected in infected cells under optimized infection condition by conventional Southern blot, suggesting a successful establishment of HBV infection after spinoculation. Finally, the parental HepG2 cells remained uninfected under HBV spinoculation, and HBV entry inhibitors targeting NTCP blocked HBV infection when cells were spinoculated, suggesting the authentic virus entry mechanism is unaltered under centrifugal inoculation. Our data suggest that spinoculation could serve as a standard protocol for enhancing the efficiency of HBV infection in vitro.     

免疫检查点分子在慢性HBV感染中对T细胞功能影响的研究进展

阻断免疫检查点分子的信号通路可以增强免疫系统功能,这在肿瘤治疗中获得了显著效果。乙型肝炎病毒(Hepatitis B virus,HBV)慢性感染的原因之一为HBV在体内通过一系列方式来减弱、抑制免疫系统的功能,从而逃避免疫识别和杀伤。HBV可以通过上调病毒特异性T细胞表面的抑制性受体来抑制T细胞活化、增殖和效应。本文总结了HBV导致的人体内T细胞上免疫检查点程序性死亡受体1(Programmed cell death protein 1,PD-1)、细胞毒T淋巴细胞相关抗原4(Cytotoxic T-lymphocyte-associated protein 4,CTLA-4)、T细胞免疫球蛋白黏蛋白3(T-cell immunoglobulin domain and mucin domain-containing molecule-3,Tim-3)、淋巴细胞活化基因3(Lymphocyte-activation gene 3,LAG-3)、T细胞免疫球蛋白和免疫受体酪氨酸抑制基序(T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain,TIGIT)的异常表达以及它们影响T细胞功能的机制,揭示了免疫检查点在治疗慢性乙肝中的良好应用前景。     

Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

Persistence of hepatitis B virus (HBV) infection requires covalently closed circular (ccc)DNA formation and amplification, which can occur via intracellular recycling of the viral polymerase-linked relaxed circular (rc) DNA genomes present in virions. Here we reveal a fundamental difference between HBV and the related duck hepatitis B virus (DHBV) in the recycling mechanism. Direct comparison of HBV and DHBV cccDNA amplification in cross-species transfection experiments showed that, in the same human cell background, DHBV but not HBV rcDNA converts efficiently into cccDNA. By characterizing the distinct forms of HBV and DHBV rcDNA accumulating in the cells we find that nuclear import, complete versus partial release from the capsid and complete versus partial removal of the covalently bound polymerase contribute to limiting HBV cccDNA formation; particularly, we identify genome region-selectively opened nuclear capsids as a putative novel HBV uncoating intermediate. However, the presence in the nucleus of around 40% of completely uncoated rcDNA that lacks most if not all of the covalently bound protein strongly suggests a major block further downstream that operates in the HBV but not DHBV recycling pathway. In summary, our results uncover an unexpected contribution of the virus to cccDNA formation that might help to better understand the persistence of HBV infection. Moreover, efficient DHBV cccDNA formation in human hepatoma cells should greatly facilitate experimental identification, and possibly inhibition, of the human cell factors involved in the process.     

Fibronectin is essential for hepatitis B virus propagation in vitro: may be a potential cellular target?

Previous studies in our laboratory strongly suggested that fibronectin was upregulated by hepatitis B virus (HBV) in HepG2.2.15 cells. Report by Budkowska A also indicated that human liver fibronectin could bind HBV in a species-restricted manner. Therefore, it is reasonable to ask whether inhibiting fibronectin expression might have anti-HBV activity and whether fibronectin might be developed as a new potential cellular target for anti-HBV drugs. By using fibronectin antisense oligonucleotide (ASODN), fibronectin antibody, and Protocatechuic aldehyde (PA), we were able to show that HBV productions in HepG2.2.15 cell culture were reduced in a dose-dependent manner by fibronectin inhibition. In addition, we found that treatment with ASODNs, fibronectin antibody, and PA did not affect HepG2.2.15 cell viability. Furthermore, we observed that fibronectin inhibition sensitized HBV to anti-HBV drugs. In summary, this study demonstrates that fibronectin is essential for HBV propagation and also provides some evidences for the potential of fibronectin as a new cellular target for HBV infection therapy.     

Hepatitis D Virus Infection of Mice Expressing Human Sodium Taurocholate Co-transporting Polypeptide

Hepatitis D virus (HDV) is the smallest virus known to infect human. About 15 million people worldwide are infected by HDV among those 240 million infected by its helper hepatitis B virus (HBV). Viral hepatitis D is considered as one of the most severe forms of human viral hepatitis. No specific antivirals are currently available to treat HDV infection and antivirals against HBV do not ameliorate hepatitis D. Liver sodium taurocholate co-transporting polypeptide (NTCP) was recently identified as a common entry receptor for HDV and HBV in cell cultures. Here we show HDV can infect mice expressing human NTCP (hNTCP-Tg). Antibodies against critical regions of HBV envelope proteins blocked HDV infection in the hNTCP-Tg mice. The infection was acute yet HDV genome replication occurred efficiently, evident by the presence of antigenome RNA and edited RNA species specifying large delta antigen in the livers of infected mice. The resolution of HDV infection appears not dependent on adaptive immune response, but might be facilitated by innate immunity. Liver RNA-seq analyses of HDV infected hNTCP-Tg and type I interferon receptor 1 (IFNα/βR1) null hNTCP-Tg mice indicated that in addition to induction of type I IFN response, HDV infection was also associated with up-regulation of novel cellular genes that may modulate HDV infection. Our work has thus proved the concept that NTCP is a functional receptor for HDV infection in vivo and established a convenient small animal model for investigation of HDV pathogenesis and evaluation of antiviral therapeutics against the early steps of infection for this important human pathogen.     

Overexpression of hepatitis B virus-binding protein, squamous cell carcinoma antigen 1, extends retention of hepatitis B virus in mouse liver

How receptors mediate the entry of hepatitis B virus （HBV） into the target liver cells is poorly understood. Recently, human squamous cell carcinoma antigen 1 （SCCA1） has been found to mediate binding and internalization of HBV to liver-derived cell lines in vitro. In this report, we investigate if SCCA1 is able to function as an HBV receptor and mediate HBV entry into mouse liver. SCCA1 transgene under the control of Rous sarcoma virus promoter was constructed in a minicircle DNA vector that was delivered to NOD/SCID mouse liver using the hydrodynamic technique. Subsequently, HBV-positive human serum was injected intravenously. We demonstrated that approximately 30% of the mouse liver cells expressed a high level of recombined SCCA1 protein for at least 37 d. The HBV surface antigen was found to persist in mouse liver for up to 17 d. Furthermore, HBV genome also persisted in mouse liver, as determined by polymerase chain reaction, for up to 17 d, and in mouse circulation for 7 d. These results suggest that SCAA1 might serve as an HBV receptor or co-receptor and play an important role in mediating HBV entry into hepatocytes, although its role in human HBV infection remains to be determined.     

Peripheral T-cell subsets in asymptomatic hepatitis B-virus carriers

To ascertain whether the abnormalities of circulating T-cell subsets in patients with hepatitis B virus (HBV)-related chronic liver diseases represent the primary immunological process or are secondary to liver disease process, peripheral T-cell subsets were analyzed by indirect immunofluorescence using monoclonal antibodies against total T cells (OKT3), T helper/inducer cells (OKT4), and T suppressor/cytotoxic cells (OKT8), in 30 asymptomatic HBV carriers without biochemical or histological evidence of liver disease, and the results were compared to 15 HBV-induced chronic active liver diseases. The results revealed that OKT4/OKT8 ratios were significantly reduced in 15 hepatitis B e antigen (HBeAg)-positive asymptomatic carriers as compared with controls, with decreased OKT4-positive cells and increased OKT8-positive cells, while T-cell subsets and ratios were normal in 15 hepatitis B e antibody (anti-HBe)-positive asymptomatic carriers. The changes of circulating T-cell subsets in 15 HBe-Ag-positive asymptomatic carriers showed no significant difference from those of 15 HBeAg-positive patients with chronic active liver diseases. These findings suggest that the deranged T-cell subsets in chronic HBV infection are not secondary to liver cell damage, but might represent the underlying immunological abnormalities which are closely related to HBeAg/anti-HBe status, and that the pathogenetic mechanism of liver cell damage in chronic HBV infection may not be simply related to circulating T-cell subsets.     

Proteomic analysis of plasma membranes isolated from undifferentiated and differentiated HepaRG cells

ABSTRACT: Liver infection with hepatitis B virus (HBV), a DNA virus of the Hepadnaviridae family, leads to severe disease, such as fibrosis, cirrhosis and hepatocellular carcinoma. The early steps of the viral life cycle are largely obscure and the host cell plasma membrane receptors are not known. HepaRG is the only proliferating cell line supporting HBV infection in vitro, following specific differentiation, allowing for investigation of new host host-cell factors involved in viral entry, within a more robust and reproducible environment. Viral infection generally begins with receptor recognition at the host cell surface, following highly specific cell-virus interactions. Most of these interactions are expected to take place at the plasma membrane of the HepaRG cells. In the present study, we used this cell line to explore changes between the plasma membrane of undifferentiated (-) and differentiated (+) cells and to identify differentially-regulated proteins or signaling networks that might potentially be involved in HBV entry. Our initial study identified a series of proteins that are differentially expressed in the plasma membrane of (-) and (+) cells and are good candidates for potential cell-virus interactions. To our knowledge, this is the first study using functional proteomics to study plasma membrane proteins from HepaRG cells, providing a platform for future experiments that will allow us to understand the cell-virus interaction and mechanism of HBV viral infection.