Next Article in Journal
Changes in Dento-Facial Morphology Induced by Wind Instruments, in Professional Musicians and Physical Exercises That Can Prevent or Improve Them—A Systematic Review
Next Article in Special Issue
Hepatitis B in Pediatric Population: Observational Retrospective Study in Romania
Previous Article in Journal
Lessons from Polyomavirus Immunofluorescence Staining of Urinary Decoy Cells
Previous Article in Special Issue
Age and Gender Trends in the Prevalence of Markers for Hepatitis E Virus Exposure in the Heterogeneous Bulgarian Population
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Life
Volume 13
Issue 7
10.3390/life13071527
life-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

Cell Culture Systems for Studying Hepatitis B and Hepatitis D Virus Infections

by
Grace Sanghee Lee
,
Michael A. Purdy
and
Youkyung Choi
*
Division of Viral Hepatitis, National Center for HIV, Viral Hepatitis, STD and TB Prevention, US Centers for Disease Control and Prevention (CDC), Atlanta, GA 30333, USA
*
Author to whom correspondence should be addressed.
Life 2023, 13(7), 1527; https://doi.org/10.3390/life13071527
Submission received: 12 June 2023 / Revised: 3 July 2023 / Accepted: 6 July 2023 / Published: 8 July 2023
(This article belongs to the Special Issue Epidemiology and Control of Hepatitis Viruses)
Browse Figure
Versions Notes

Abstract

:
The hepatitis B virus (HBV) and hepatitis D virus (HDV) infections cause liver disease, including hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HBV infection remains a major global health problem. In 2019, 296 million people were living with chronic hepatitis B and about 5% of them were co-infected with HDV. In vitro cell culture systems are instrumental in the development of therapeutic targets. Cell culture systems contribute to identifying molecular mechanisms for HBV and HDV propagation, finding drug targets for antiviral therapies, and testing antiviral agents. Current HBV therapeutics, such as nucleoside analogs, effectively suppress viral replication but are not curative. Additionally, no effective treatment for HDV infection is currently available. Therefore, there is an urgent need to develop therapies to treat both viral infections. A robust in vitro cell culture system supporting HBV and HDV infections (HBV/HDV) is a critical prerequisite to studying HBV/HDV pathogenesis, the complete life cycle of HBV/HDV infections, and consequently identifying new therapeutics. However, the lack of an efficient cell culture system hampers the development of novel antiviral strategies for HBV/HDV infections. In vitro cell culture models have evolved with significant improvements over several decades. Recently, the development of the HepG2-NTCP sec+ cell line, expressing the sodium taurocholate co-transporting polypeptide receptor (NTCP) and self-assembling co-cultured primary human hepatocytes (SACC-PHHs) has opened new perspectives for a better understanding of HBV and HDV lifecycles and the development of specific antiviral drug targets against HBV/HDV infections. We address various cell culture systems along with different cell lines and how these cell culture systems can be used to provide better tools for HBV and HDV studies.

1. Introduction

The hepatitis B virus (HBV) infection causes acute and chronic liver disease and remains a major global health problem [1]. Chronic HBV infection is characterized by the persistence of covalently closed circular HBV DNA (cccDNA) in the nucleus of infected hepatocytes and may lead to fibrosis, cirrhosis [2,3], and hepatocellular carcinoma (HCC) [4]. Current therapeutics for chronic hepatitis B effectively block reverse transcriptase to suppress HBV DNA synthesis but do not eliminate the cccDNA or hepatitis B surface antigen (HBsAg) [5,6]. The molecular mechanisms by which HBV establishes persistent infection remain to be elucidated, but the stability of the HBV genome in the nucleus of hepatocytes is believed to be a key mechanism of HBV’s chronicity [4,7].
The hepatitis D virus (HDV, also known as hepatitis delta), a satellite virus of HBV, is highly pathogenic and requires HBsAg for its propagation and infectivity [8]. There are two major specific patterns of HDV infection. One is the simultaneous infection of a healthy individual with HBV, called a “co-infection”, and the other is “super-infection”, which is the subsequent HDV infection of a patient with chronic hepatitis B [9]. A lack of understanding of virus–host interactions limit the development of efficient curative treatment for HBV/HDV infection. Therefore, a robust cell model could provide useful tools to better understand HBV and HDV life cycles, replication, and interaction with the host. In this review, in vitro, hepatocyte culture systems to support the HBV and HDV lifecycles are discussed to highlight the advantages and the limitations of current systems.

2. HBV and HDV Biology

2.1. Structure and Genome Organization

HBV is a prototype member of the Hepadnaviridae family: an enveloped virus with a 30–42 nm diameter. Ten HBV genotypes (A to J) have been identified [10]. The hepatitis B virion has a spherical, double-shelled structure consisting of lipid-embedded small (S), middle (M), and large (L) hepatitis B surface proteins (HBsAg) that surround an icosahedral inner nucleocapsid composed of the hepatitis B core antigen (HBcAg) [11] (Figure 1). L-HBsAg contains three domains, including pre-S1, pre-S2, and S [12]. M-HBsAg lacks the pre-S1 domain, while S-HBsAg consists of only the S region, which forms a disulfide crosslinked dimer [13]. Pre-S1 and pre-S2 are essential components of the protein envelope of the complete hepatitis B virion [14]. Pre-S1 contains the receptor binding region for HBV [15] and plays important roles in HBV assembly, infection, replication, HBsAg secretion from hepatocytes, and the activation of host immune responses [16,17]. Mutations within the pre-S (pre-S1 and pre-S2) gene can cause immune escape, which affects HBsAg expression and liver disease progression [18]. Point mutations in the start codon of preS2 inhibit the M protein expression completely [19]. In addition, pre-S mutations, including deletions in the pre-S1 and/or pre-S2 region and point mutations, can also be found in patients with chronic hepatitis B (CHB) [20]. Pre-S1 and/or pre-S2 proteins are detected in both HBV-DNA-negative chronic HBsAg carriers in the inactive HbsAg carrier phase and in HBV DNA positive sera in patients in the immune tolerance phase [21], suggesting the presence of pre-S1 and pre-S2 proteins in the serum and liver, which does not reflect the presence of active HBV replication or active liver disease [22,23]. The expression of the HBV core gene produces the core antigen (HBcAg) and the e antigen (HBeAg), which were initiated from different start codons in the same open reading frame (ORF), resulting in two unique homo-dimeric proteins with different structures and functions [24]. HBcAg or HBeAg levels were correlated with HBV DNA [25,26]. Additionally, the nucleocapsid structure of HBcAg is an indicator of viral replication and is the most critical viral factor for HBV clearance in vivo [27]. HBcAg is also associated with HBV-specific T-cell responses [28]. HBcAg is mainly localized in the cytoplasm and is particulate because it is neither secreted nor circulated in the blood. HBeAg is secreted into the blood stream [29,30] and is located between the nucleocapsid core and the lipid envelope. The presence of HBeAg is an indicator of active HBV replication and infection [31,32,33].
HBV has a partially double-stranded and relaxed circular DNA (rcDNA) genome of 3.2 kb whose 5′ ends of the minus strand are covalently attached to the viral polymerase [34]. HBV X protein (HBx) is a multifunctional viral protein that regulates transcriptional activation, cell cycle progression, DNA repair, protein degradation, and several signaling pathways [35]. HBsAg assists with the packaging of the HDV ribonucleoprotein (RNP), leading to HDV assembly and the cellular egress of the virus particle [36]. L-HBsAg, but not M-HBsAg, is essential for HBV and HDV infection [37].
HDV is an enveloped small satellite virus with a 35–37 nm diameter and belongs to the genus, Deltaviridae from the family Kolmioviridae [10,38]. It is the smallest known virus to infect humans and requires HBsAg to become virulent [39]. HDV is currently classified into eight species (HDV-1 to HDV-8) [10]. The inner nucleocapsid of the virus contains a negative sense single-stranded circular RNA of 1.7 kb and hepatitis delta antigen (HDAg), which is the only known HDV protein [40] and modulates viral replication through interactions with cellular DNA-dependent RNA polymerases and other host factors [41,42]. There are two forms of HDAg: one is 24 kDa small HDAg (S-HDAg) which participates in virus replication, and the other is 27 kDa large HDAg (L-HDAg) involving virion assembly [43]. HDV is a defective virus that is surrounded by three forms of HBsAg (L, M, and S) and host lipids. Both S- and L-HBsAgs are essential for HDV viral assembly [44], virion packaging, and virus infectivity [37,39,45]. The pre-S1 domain of L-HBsAg is required for HDV cell entry by binding to the sodium taurocholate co-transporting polypeptide (NTCP) receptor, indicating that the viral entry pathways of both HBV and HDV are highly similar. While L-HBsAg is not required for HDV assembly and release, it is essential for HDV infectivity [37]. However, the presence of pre-S proteins and HBsAg in the liver and serum does not indicate the replication of HBV, HDV, and secretion [22,46], suggesting that HDV replication occurs independently from HBV. The HDV genome does not encode its own polymerase but utilizes the host RNA polymerase II the viral replication, while HBV has its own viral polymerase that can be targeted by specific inhibitors [47]. It is wort noting that HDV is the only human viral pathogen that uses the host polymerase [48]. Therefore, HBV therapies based on inhibiting the HBV polymerase cannot directly target HDV infection, which hampers the development of HDV therapeutic targets.

2.2. Transmission and Life Cycle

The transmission of both HBV and HDV occurs parenterally through infectious blood or body fluids [49]. While HDV perinatal transmission is rare, HBV can be transmitted from an infected pregnant person to the newborn during pregnancy and childbirth. Thus, perinatal transmission is one of the major sources of HBV transmission [50,51,52]. Since HBV and HDV share the same surface protein (HBsAg) on their virions, the life cycle of HDV is believed to be very similar to that of HBV [53,54,55]. The replication of both HBV and HDV occurs primarily in hepatocytes [40,56,57]. To initiate viral entry and replication, the virions of HBV and HDV bind to the plasma membrane of human hepatocytes through cell-surface factors such as heparan sulfate proteoglycans (HSPGs) [58]. The entry pathway of HBV and HDV into the hepatocytes has not been fully delineated, but HBsAg, especially through the pre-S1 region, plays an essential role in the interaction with the hepatocyte plasma membrane [59,60]. A study identified that the sequence of pre-S1 modulates viral infectivity, where both HBV and HDV infections are dependent on the presence of L-HBsAg [61]. The virus internalizes into host hepatocytes by membrane fusion in an endocytosis-dependent manner [62,63,64]; however, the detailed entry mechanisms of HBV and HDV remain to be determined. HBV and HDV enter hepatocytes through NCTP: a multiple transmembrane transporter at the basolateral membrane of primary hepatocytes. The endocytic transfer of viral particles into the cytoplasm is followed by the release of HBV nucleocapsid and HDV ribonucleoprotein (RNP) complexes from endocytic vesicles, which are subsequently destined to traffic the nucleus [65,66,67]. In the nucleus, the partially double-stranded and relaxed circular DNA (rcDNA) of HBV is converted into cccDNA, which acts as the template for transcription [68,69], while HDV RNP is transported to the nucleus and releases the viral genome, which serves as the template for the transcription of HDV mRNA [40,70,71,72].

3. Cell Culture Systems for Studying HBV and HDV

Over the last two decades, a series of in vitro cell culture systems have been established to study HBV/HDV lifecycles or host viral immune responses. However, information on the host responses to HBV and HDV infection is limited, and efficient curative treatment for HBV/HDV infections is not available. Restricted host and tissue tropism may be factors behind the lack of robust in vitro cell culture systems for HBV and HDV infection [73]. In vitro cell culture systems have limitations, including modified cell morphology, the lack of an extracellular matrix, deficient accessory cells, the atypical expression of liver enzymes, a lack of proper cell-to-cell communication, short viral infection period, and rapid dedifferentiation (rapid loss of hepatic functions) following isolation and plating [74]. In addition, the innate immune responses against HBV and HDV induce different hepatocyte gene expression profiles [75,76,77,78,79]. The efficient amplification and secretion of progeny viruses are difficult to investigate using cell culture systems [80,81]. The advantages and disadvantages of different cell culture systems in the studies of HBV/HDV infection are discussed below.

3.1. Primary Human Hepatocytes

Primary human hepatocytes (PHHs) are susceptible to HBV/HDV infection [74]. PHHs are used to evaluate hepatic metabolism, drug–drug interactions, and drug toxicity in vitro. They have become the gold standard for a hepatic in vitro culture model [74,82]. PHHs have long been the exclusive hosts of various hepatotropic pathogens, including HBV, HCV, HDV, and Plasmodium parasites [83]. PHHs support the complete life cycle of HBV and HDV infections and exhibit normal hepatic functions such as hepatocyte polarization and bile production [84]. PHHs possess various hepatocyte-specific host factors and exhibit a fully functional innate immune system in response to the infection [76,78,85]. Therefore, PHHs are the most physiologically relevant in vitro culture system for HBV/HDV infection [86]. Notably, NTCP has been identified as the cellular receptor of HBV entry, and HBV particles activated Toll-like receptor signaling upon the infection of PHHs [55,87].
The limitations of PHHs for the study of HBV/HDV infection include the limited availability of high-quality donors and life-span [88], variable susceptibility to HBV/HDV infection, the loss of their differentiation functions after plating [74], difficult to manage in culture conditions because of no proliferation in the culture, variable reproducibility between batches [89], and low infection efficiency because of rapid dedifferentiation in the culture [74]. When PHH is co-cultured with nonparenchymal cells, the de-differentiation process is delayed, which prevents its loss of susceptibility to HBV [90,91]. Persistent HBV infections in PHHs are limited to high-quality hepatocyte donor lots, and replication lasts only for 14–19 days post-infection [92]. In microscale conditions, persistent HBV infection is established for 30 days in self-assembling co-cultured primary human hepatocytes (SACC-PHHs) [93]. Additionally, susceptibility to HBV infection in SACC-PHHs is not donor-dependent and can be established without suppressing innate immune signaling where SACC-PHHs persistently support both HBV/HDV coinfection and superinfection for up to 28 days in a micro-scalable system [94].
The transcriptomic analysis in traditional PHH culture systems is limited due to rapid de-differentiation and the subsequent loss of infection. However, hepatocytes in SACC-PHHs remain a mature hepatic phenotype regardless of the HBV mono and HBV/HDV co-infection condition [94]. The findings of differential gene expressions indicate no significant differences in genes that are involved in innate immune activation in HBV mono-infection and HBV/HDV co-infection [94], whereas the HDV infection induces the strong activation of IFN-β and IFN-λ in PHHs [95]. PHHs are susceptible to HBV and HDV infection, but limited availability and technical difficulties restrict their use in the HBV/HDV infection model.

3.2. HepaRG

The HepaRG cell line, derived from a hepatitis C virus (HCV)-induced liver tumor, is a well-established hepatic cell model [96]. The HepaRG cell line is considered an alternative to PHHs because this cell line expresses transcripts of various nuclear receptors (the aryl hydrocarbon receptor, pregnane X receptor, constitutive androstane receptor, and peroxisome proliferator-activated receptor alpha) and liver-specific enzymes like the major cytochrome P450 (CYP1A2, 2C9, 2D6, 2E1, 3A4) which plays a pivotal role in the detoxification of xenobiotics, cellular metabolism, and homeostasis [97]. Other hepatoma-derived cells, such as Huh7 and HepG2, do not express high levels of CYP450s like HepaRG cells [98,99]. In addition, Phase I and Phase II xenobiotic metabolizing enzymes and membrane transporters are expressed in HepaRG cells, which is comparable to PHHs [99,100]. Thus, HepaRG may have better hepatic functions than other hepatoma cell lines. HBV-infected HepaRG cells produce infectious HBV particles for more than 100 days in a differentiated state [101]. Various CYP450s expressed in differentiated HepaRG cells make them more comparable to cultured PHHs [102,103]. A caveolin-1, one of the plasma membrane components involved in the endocytosis of different ligands and signaling pathways within the cells, is required for productive HBV infection in HepaRG cells [104]. In HepaRG-NTCP cells, HBV evades the induction of interferon (IFN) and the antiviral effects of IFN-stimulated genes (ISG) [105], while HDV efficiently induces IFN-β and IFN-λ responses in differentiated HepaRG cells [79]. HDV replication is recognized by melanoma differentiation-associated protein 5 (MDA5) and induces IFN-β/λ responses but is also insensitive to the IFN-activated stage, where MDA5 depletion has little effect on HDV replication despite dampening the HDV-induced IFN response [95]. Because of these features, HepaRG cells can be used for antiviral drug metabolism and its evaluation [106,107,108].
Despite these extensive research efforts, HBV/HDV infection in HepaRG cells has not been widely studied because HBV infection in HepaRG cells is limited or there is no cell-to-cell spreading. Additionally, HBV infection appears to be a slow process where viral replication starts at around 8 post-infection days and reaches a maximum at day 13 [96,101]. In addition, the conversion of the input relaxed circular viral DNA into cccDNA is inefficient; however, no further amplification of cccDNA has been found to occur in differentiated HepaRG cells [101]. To improve these limitations, dimethyl sulfoxide (DMSO) can be used for the differentiation of HepaRG cells into hepatocyte-like cells where susceptibility to HBV infection is closely correlated to the differentiation status of HepaRG cell lines [96]. As an alternative to DMSO, forskolin promotes functional polarization and differentiation by increasing hepatic markers like cytochrome P-450 [109]. A cocktail of five chemicals (5C-medium), Forskolin (an adenylate cyclase inhibitor) combined with SB431542 (a TGF-β inhibitor), IWP2 (a Wnt inhibitor), DAPT (a Notch inhibitor) and LDN193189 (an inhibitor of bone morphogenetic protein, BMP), accelerate the differentiation of HepaRG cells and have been used for maintaining the differentiated PHH characteristics of HepaRG cells [110]. However, differentiation using the 5C cocktail in HepaRG cells does not increase cccDNA levels or subsequent HBV replication markers, although it improves the efficiency of HDV infection [111].

3.3. Huh7

The Huh7 cell is a permanent cell line that is derived from hepatocellular carcinoma and is used as an experimental substitute for primary hepatocytes [112]. However, Huh7 cells only partially mimic normal hepatocytes due to their poor polarization [113]. The pre-S1 domain of the HBV large envelope protein is a key determinant for receptor(s) binding; however, the Huh7 cell line has not been shown to have detectable amounts of the HBV cellular receptor [114]. After identifying hNTCP as a functional receptor for HBV and HDV infection, NTCP-overexpressing Huh7 cells were permissible for HBV and HDV infection and were used for the study of the HBV/HDV lifecycle and specifically for viral entry [115]. Another stable cell line, Huh7-END (Huh7-HDV-Env-NTCP clone B1), also supports the continuous and high-level production of HDV particles and supports the virus spread to co-cultured cells [116]. Since Huh7-END expresses NTCP, it is susceptible to de novo HDV entry. Thus, HuH7-END cells could be used for the screening of antiviral drugs targeting HDV replication.
Most in vitro cell culture systems for HBV infection except PHHs require DMSO, but a study has reported that DMSO is not required for HBV infection in Huh7.5-NTCP cells: a derivative of the HuH7 cell line [117]. DMSO supplementation induces cell growth arrest [118], an alteration in protein expression [119], and cytotoxicity [120], resulting in the failure to restore many liver functions. These studies suggest that the elimination of DMSO treatment in the cell culture system could improve HBV infection.
The Huh7 cell line is permissive for HBV replication and viral particle formation but not to events relating to viral uncoating and replication due to a lack of unidentified host factor(s) [121]. In mammalian species, pattern recognition receptors (PRRs) such as the retinoic acid-inducible gene I (RIG-1), MDA5, and Toll-like receptors (TLRs) recognize RNA viruses and play an essential role in the activation of innate immune responses. HDV infection induces the type I interferon response in infected cells; however, these responses are not able to suppress HDV replication or spread [122]. Another study showed that IFN-α-induced intracellular signaling is impaired in HDV-transfected Huh7 cells where HDV subverts the effect of the IFN-alpha by blocking tyrosine kinase 2 (Tyk2) activation and results in the selective impairment of activation and the translocation of the signal transducer and activator of transcription 1 (STAT1) and signal transducer and activator of transcription 2 (STAT2) to the nucleus [123]. The interference of IFN-α signaling by HDV could be an important mechanism of viral persistence and treatment resistance.

3.4. HepG2 and HepG2-NTCP

HepG2 cells were isolated from a human hepatocellular carcinoma. HepG2 cell lines are widely used in drug metabolism and hepatotoxicity studies [55,63] even if HepG2 cells exhibit much lower expression levels of many drug-metabolizing enzymes such as cytochrome P450 compared to PHHs [124,125,126]. Two HepG2-derived stably transfected cell lines, HepG2.2.15 [127] and HepAD38 [128], were used to produce cell culture-derived HBV, accessing the late stage of the virus life cycle and antiviral research [129,130,131]. The exogenous expression of NTCP in HepG2 cells supported the entire HBV life cycle and viral spread [121,132]. A commercial human inducible pluripotent stem cell (iPSC)-derived hepatocyte maintenance medium (HMM) was shown to enhance HBV infection and NTCP expression in HepG2-NTCP cells [133]. With features like efficient viral infection and high reproducibility experimental results, HepG2-NTCP cells are a surrogate model for hepatocytes [55,134].
A comparison of gene expression profiles in primary human hepatocytes, including hepatocellular cell lines such as HepG2 and Huh7, and human liver tissue have indicated that hepatocellular cell lines have a highly differentiated nature of hepatocytes compared to liver tissues [125,135,136]. Additionally, NTCP-overexpressed HepG2, Huh7, and undifferentiated HepaRG cells have substantially different infection efficiencies for HBV and HDV. HepG2-NTCP cells appear to be more susceptible to HBV than Huh7-NTCP cells, whereas Huh7-NTCP cells and HepaRG have better infection efficiencies than HDV infection [121]. These studies suggest that additional host factors may be needed for optimal virus infection. HMM activates the cytomegalovirus immediate-early (IE) promoter, which is induced by NTCP expression in the HepG2-NTCP cells and leads to the upregulation of several metabolic pathways. Microarray analysis using HDV- and HBV-infected HepG2-NTCP cells and PHH found that HDV, but not HBV infection, strongly activated a broad range of interferon-stimulated genes (ISGs): IFN-β and IFN-γ; however, HDV replication was mostly insensitive to innate immune responses mediated by MDA5 and was not directly affected by exogenous IFNs after the establishment of the infection [95].
The HepG2-NTCP cell culture system clearly provides useful tools to better understand the life cycles of HBV and HDV infection. However, there are some limitations of HBV and/or HDV infection in HepG2-NTCP cells. Viral replication is limited due to a lack of miRNA-122 in HepG2 because miRNA-122 is the most abundant liver-specific miRNA and plays key roles in liver development and hepatic function. The loss of miRNA-122 was found in liver cancer in HBV-infected livers [137]. The extra use of polyethylene glycol (PEG) and DMSO could restrict the study of HBV/HDV infection pathways by inhibiting the amplification of the viral genome and viral progeny production [138,139,140]. The elimination of the extra usage of PEG and DMSO could improve infection efficiency [141,142,143]. These improvements are desirable to more closely mimic physiological conditions.

3.5. NCTP-Expressed Hepatoma Derived HepG2 Cell Lines

Recently developed HepG2-NTCP sec+ cells support the complete HBV life cycle, long-term viral spread, and amplification of HBV derived from patients’ serum samples; however, it does not induce significant gene expression changes in HepG2-NTCP sec+ [144]. However, HepG2-NTCP sec+ cells have a short-distance route for HBV spread to neighboring cells, causing HBV-infected cell clusters and requiring high viral titer inoculum (up to 5000 GEq/mL) as well as PEG to increase infectivity. Considering the practical disadvantages of HepG2-NTCP cells, the HepG2-NTCP-A3/C2 subclone has relatively increased viral production under PEG-free conditions and showed an improved in vitro HBV infection system [145]. Additionally, hepatocyte nuclear factor 4α and the NTCP-expressing hepatoma cell line, BEL7404, have been permissive for HBV replication and susceptible to HBV infection [146]. Nevertheless, more cell culture models with increased susceptibility to HBV infection need to be identified and studied.

3.6. The 3D Culture

The in vitro culturing and maintenance of hepatocytes are difficult because they are rapidly losing their cuboidal morphology and liver-specific functions [147,148]. The elongated spindle morphology of hepatocytes has been observed in 2D-cultured PHH monolayers [149,150]. To maintain an intact morphology, 3D-cultured PHHs have developed a spheroid morphology [151]. The most common 3D culture method is placing PHHs on a single-layer collagen matrix; however, basic liver functions decrease in this cell culture system after a week [152]. To prolong and enhance hepatic functions in vitro, a 3D cell culture was developed by fabricating PHH microtissues using droplet microfluidics encapsulated with fibroblasts [153]. This new system displayed the stable expression of hepatocyte genes and maintained functional liver-specific genes for a month or longer. In addition, lower levels of apoptotic markers were detected in 3D-cultured PHHs compared to 2D-cultured PHHs [154,155]. The 3D cultures of Huh7 cells exhibited improved hepatocyte differentiation [156,157]. The 3D-cultured PHHs do not need either PEG or DMSO for HBV infection [86], although no differences were seen in cytochrome P450 enzyme levels between 2D- and 3D-cultured PHHs [155]. However, maintaining a natural hepatic environment and liver-specific functions such as the expression of albumen, transferrin, apoptotic-related markers, and metabolizing enzymes in cell culture systems is still greatly desired.

4. Conclusions and Future Developments

A robust in vitro cell culture system supporting HBV and/or HDV infection is essential to study life in the cycles of HBV and/or HDV and to further develop new therapeutic approaches. The development of cell culture systems using PHH, HepaRG, Huh7, and HepG2 cells has significantly contributed to studying the molecular mechanisms that underlie HBV and HDV infections. The discovery of NTCP as an entry receptor of HBV and HDV has led to the establishment of in vitro cell culture systems for HBV and HDV infection [55,121]. Despite these advances, HBV and HDV research is still limited because of the lack of experimental models supporting native HBV and/or HDV infections (Table 1). Culturing hepatocytes supporting HBV/HDV replication and maturation has limitations such as a lack of complete HBV/HDV life cycles, low infection efficiency, no viral spread, the use of polyethylene glycol (PEG) during entry to improve glycosaminoglycan-dependent binding [142], the use of DMSO to enhance HBV infection [143], and low cccDNA production rates [158,159]. The ultimate goal of in vitro cell culture study is to establish standardized and reproducible conditions for HBV/HDV infection, the better characterization of viral progeny, and increased cccDNA production, which is needed for the development of a cure for chronic hepatitis B infection.

Author Contributions

Conceptualization, Y.C. and M.A.P.; data curation, G.S.L. and Y.C.; writing—original draft preparation, G.S.L. and Y.C.; writing—review and editing, G.S.L., M.A.P. and Y.C.; visualization, G.S.L.; supervision, M.A.P.; project administration, Y.C. and M.A.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the US Centers for Disease Control and Prevention.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are included in this article.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. WHO. Hepatitits B Fact Sheet. Available online: https://www.who.int/news-room/fact-sheets/detail/hepatitis-b#:~:text=WHO%20estimates%20that%20296%20million,carcinoma%20(primary%20liver%20cancer) (accessed on 12 June 2023).
  2. Qi, Z.; Li, G.; Hu, H.; Yang, C.; Zhang, X.; Leng, Q.; Xie, Y.; Yu, D.; Zhang, X.; Gao, Y.; et al. Recombinant Covalently Closed Circular Hepatitis B Virus DNA Induces Prolonged Viral Persistence in Immunocompetent Mice. J. Virol. 2014, 88, 8045–8056. [Google Scholar] [CrossRef]
  3. Ko, C.; Chakraborty, A.; Chou, W.-M.; Hasreiter, J.; Wettengel, J.M.; Stadler, D.; Bester, R.; Asen, T.; Zhang, K.; Wisskirchen, K.; et al. Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels. J. Hepatol. 2018, 69, 1231–1241. [Google Scholar] [CrossRef]
  4. Kar, A.; Samanta, A.; Mukherjee, S.; Barik, S.; Biswas, A. The HBV web: An insight into molecular interactomes between the hepatitis B virus and its host en route to hepatocellular carcinoma. J. Med. Virol. 2023, 95, e28436. [Google Scholar] [CrossRef]
  5. Werle-Lapostolle, B.; Bowden, S.; Locarnini, S.; Wursthorn, K.; Petersen, J.; Lau, G.; Trepo, C.; Marcellin, P.; Goodman, Z.; Delaney, W.E., IV; et al. Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy. Gastroenterology 2004, 126, 1750–1758. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Reijnders, J.G.; Rijckborst, V.; Sonneveld, M.J.; Scherbeijn, S.M.; Boucher, C.A.; Hansen, B.E.; Janssen, H.L. Kinetics of hepatitis B surface antigen differ between treatment with peginterferon and entecavir. J. Hepatol. 2011, 54, 449–454. [Google Scholar] [CrossRef] [PubMed]
  7. Dandri, M.; Petersen, J. Mechanism of Hepatitis B Virus Persistence in Hepatocytes and Its Carcinogenic Potential. Clin. Infect. Dis. 2016, 62, S281–S288. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  8. Stockdale, A.J. Hepatitis D. In Comprehensive Guide to Hepatitis Advances; Elsevier: Amsterdam, The Netherlands, 2023; pp. 281–307. [Google Scholar]
  9. Smedile, A.; Dentico, P.; Zanetti, A.; Sagnelli, E.; Nordenfelt, E.; Actis, G.C.; Rizzetto, M. Infection with the Delta Agent in Chronic HBsAg Carriers. Gastroenterology 1981, 81, 992–997. [Google Scholar] [CrossRef] [PubMed]
  10. Magnius, L.; Taylor, J.; Mason, W.S.; Sureau, C.; Dény, P.; Norder, H.; ICTV Report Consortium. ICTV Virus Taxonomy Profile: Deltavirus. J. Gen. Virol. 2018, 99, 1565–1566. [Google Scholar] [CrossRef] [PubMed]
  11. Dryden, K.A.; Wieland, S.F.; Whitten-Bauer, C.; Gerin, J.L.; Chisari, F.V.; Yeager, M. Native Hepatitis B Virions and Capsids Visualized by Electron Cryomicroscopy. Mol. Cell 2006, 22, 843–850. [Google Scholar] [CrossRef] [PubMed]
  12. Neurath, A.R.; Kent, S.B.H.; Strick, N.; Taylor, P.; Stevens, C.E. Hepatitis B virus contains pre-S gene-encoded domains. Nature 1985, 315, 154–156. [Google Scholar] [CrossRef]
  13. Venkatakrishnan, B.; Zlotnick, A. The Structural Biology of Hepatitis B Virus: Form and Function. Annu. Rev. Virol. 2016, 3, 429–451. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. Le Seyec, J.; Chouteau, P.; Cannie, I.; Guguen-Guillouzo, C.; Gripon, P. Role of the Pre-S2 Domain of the Large Envelope Protein in Hepatitis B Virus Assembly and Infectivity. J. Virol. 1998, 72, 5573–5578. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  15. Gripon, P.; Cannie, I.; Urban, S. Efficient Inhibition of Hepatitis B Virus Infection by Acylated Peptides Derived from the Large Viral Surface Protein. J. Virol. 2005, 79, 1613–1622. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Blanchet, M.; Sureau, C. Infectivity Determinants of the Hepatitis B Virus Pre-S Domain Are Confined to the N-Terminal 75 Amino Acid Residues. J. Virol. 2007, 81, 5841–5849. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  17. Schulze, A.; Schieck, A.; Ni, Y.; Mier, W.; Urban, S. Fine Mapping of Pre-S Sequence Requirements for Hepatitis B Virus Large Envelope Protein-Mediated Receptor Interaction. J. Virol. 2010, 84, 1989–2000. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  18. Kao, J.-H.; Chen, P.-J.; Chen, D.-S. Recent Advances in the Research of Hepatitis B Virus-Related Hepatocellular Carcinoma: Epidemiologic and molecular biological aspects. Adv. Cancer Res. 2010, 108, 21–72. [Google Scholar] [CrossRef]
  19. Chen, B.; Liu, C.; Jow, G.; Chen, P.; Kao, J.; Chen, D. High Prevalence and Mapping of Pre-S Deletion in Hepatitis B Virus Carriers with Progressive Liver Diseases. Gastroenterology 2006, 130, 1153–1168. [Google Scholar] [CrossRef]
  20. Pollicino, T.; Cacciola, I.; Saffioti, F.; Raimondo, G. Hepatitis B virus PreS/S gene variants: Pathobiology and clinical implications. J. Hepatol. 2014, 61, 408–417. [Google Scholar] [CrossRef] [Green Version]
  21. Yim, H.J.; Lok, A.S.-F. Natural history of chronic hepatitis B virus infection: What we knew in 1981 and what we know in 2005. Hepatology 2006, 43, S173–S181. [Google Scholar] [CrossRef] [Green Version]
  22. van Ditzhuijsen, T.J.M.; Kuijpers, L.P.C.; Koens, M.J.; Rijntjes, P.J.M.; van Loon, A.M.; Yap, S.H. Hepatitis B pre-S1 and pre-S2 proteins: Clinical significance and relation to hepatitis B virus DNA. J. Med. Virol. 1990, 32, 87–91. [Google Scholar] [CrossRef]
  23. Lian, M.; Zhou, X.; Wei, L.; Qiu, S.; Zhou, T.; Li, L.; Gu, X.; Luo, M.; Zheng, X. Serum levels of preS antigen (HBpreSAg) in chronic hepatitis B virus infected patients. Virol. J. 2007, 4, 93. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Miyanohara, A.; Imamura, T.; Araki, M.; Sugawara, K.; Ohtomo, N.; Matsubara, K. Expression of hepatitis B virus core antigen gene in Saccharomyces cerevisiae: Synthesis of two polypeptides translated from different initiation codons. J. Virol. 1986, 59, 176–180. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Mixson-Hayden, T.; Purdy, M.A.; Ganova-Raeva, L.; McGovern, D.; Forbi, J.C.; Kamili, S. Evaluation of performance characteristics of hepatitis B e antigen serologic assays. J. Clin. Virol. 2018, 109, 22–28. [Google Scholar] [CrossRef]
  26. Kimura, T.; Rokuhara, A.; Matsumoto, A.; Yagi, S.; Tanaka, E.; Kiyosawa, K.; Maki, N. New Enzyme Immunoassay for Detection of Hepatitis B Virus Core Antigen (HBcAg) and Relation between Levels of HBcAg and HBV DNA. J. Clin. Microbiol. 2003, 41, 1901–1906. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Lin, Y.-J.; Wu, H.-L.; Chen, D.-S.; Chen, P.-J. Hepatitis B Virus Nucleocapsid but Not Free Core Antigen Controls Viral Clearance in Mice. J. Virol. 2012, 86, 9266–9273. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  28. Aliabadi, E.; Urbanek-Quaing, M.; Maasoumy, B.; Bremer, B.; Grasshoff, M.; Li, Y.; Niehaus, C.E.; Wedemeyer, H.; Kraft, A.R.M.; Cornberg, M. Impact of HBsAg and HBcrAg levels on phenotype and function of HBV-specific T cells in patients with chronic hepatitis B virus infection. Gut 2021, 71, 2300–2312. [Google Scholar] [CrossRef]
  29. Kramvis, A.; Kostaki, E.-G.; Hatzakis, A.; Paraskevis, D. Immunomodulatory Function of HBeAg Related to Short-Sighted Evolution, Transmissibility, and Clinical Manifestation of Hepatitis B Virus. Front. Microbiol. 2018, 9, 2521. [Google Scholar] [CrossRef] [Green Version]
  30. Carlier, D.; Jean-Jean, O.; Rossignol, J.-M. Characterization and biosynthesis of the woodchuck hepatitis virus e antigen. J. Gen. Virol. 1994, 75, 171–175. [Google Scholar] [CrossRef]
  31. Liaw, Y.-F. HBeAg seroconversion as an important end point in the treatment of chronic hepatitis B. Hepatol. Int. 2009, 3, 425–433. [Google Scholar] [CrossRef] [Green Version]
  32. Hadziyannis, S.J.; Lieberman, H.M.; Karvountzis, G.G.; Shafritz, D.A. Analysis of Liver Disease, Nuclear HBcAg, Viral Replication, and Hepatitis B Virus DNA in Liver and Serum of HBcAg vs. Anti-HBe Positive Carriers of Hepatitis B Virus. Hepatology 1983, 3, 656–662. [Google Scholar] [CrossRef]
  33. Liang, T.J.; Ghany, M. Hepatitis B e Antigen—The Dangerous Endgame of Hepatitis B. N. Engl. J. Med. 2002, 347, 208–210. [Google Scholar] [CrossRef] [PubMed]
  34. Guo, H.; Jiang, D.; Zhou, T.; Cuconati, A.; Block, T.M.; Guo, J.-T. Characterization of the Intracellular Deproteinized Relaxed Circular DNA of Hepatitis B Virus: An Intermediate of Covalently Closed Circular DNA Formation. J. Virol. 2007, 81, 12472–12484. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Belloni, L.; Pollicino, T.; De Nicola, F.; Guerrieri, F.; Raffa, G.; Fanciulli, M.; Raimondo, G.; Levrero, M. Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function. Proc. Natl. Acad. Sci. USA 2009, 106, 19975–19979. [Google Scholar] [CrossRef] [PubMed]
  36. Freitas, N.; Cunha, C.; Menne, S.; Gudima, S.O. Envelope Proteins Derived from Naturally Integrated Hepatitis B Virus DNA Support Assembly and Release of Infectious Hepatitis Delta Virus Particles. J. Virol. 2014, 88, 5742–5754. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  37. Sureau, C.; Guerra, B.; Lanford, R.E. Role of the large hepatitis B virus envelope protein in infectivity of the hepatitis delta virion. J. Virol. 1993, 67, 366–372. [Google Scholar] [CrossRef] [Green Version]
  38. Walker, P.J.; Siddell, S.G.; Lefkowitz, E.J.; Mushegian, A.R.; Adriaenssens, E.M.; Dempsey, D.M.; Dutilh, B.E.; Harrach, B.; Harrison, R.L.; Hendrickson, R.C.; et al. Changes to virus taxonomy and the Statutes ratified by the International Committee on Taxonomy of Viruses (2020). Arch. Virol. 2020, 165, 2737–2748. [Google Scholar] [CrossRef]
  39. Rizzetto, M.; Hoyer, B.; Canese, M.G.; Shih, J.W.; Purcell, R.H.; Gerin, J.L. delta Agent: Association of delta antigen with hepatitis B surface antigen and RNA in serum of delta-infected chimpanzees. Proc. Natl. Acad. Sci. USA 1980, 77, 6124–6128. [Google Scholar] [CrossRef]
  40. Chen, P.J.; Kalpana, G.; Goldberg, J.; Mason, W.; Werner, B.; Gerin, J.; Taylor, J. Structure and replication of the genome of the hepatitis delta virus. Proc. Natl. Acad. Sci. USA 1986, 83, 8774–8778. [Google Scholar] [CrossRef]
  41. Lai, M.M.; Kobayashi, M.; Koike, K. Molecular biology of hepatitis delta virus. Tanpakushitsu Kakusan Koso 1990, 35, 2108–2116. [Google Scholar] [CrossRef]
  42. Taylor, J.M. Hepatitis delta virus. Virology 2006, 344, 71–76. [Google Scholar] [CrossRef] [Green Version]
  43. Tavanez, J.P.; Cunha, C.; Silva, M.C.; David, E.; Monjardino, J.; Carmo-Fonseca, M. Hepatitis delta virus ribonucleoproteins shuttle between the nucleus and the cytoplasm. RNA 2002, 8, 637–646. [Google Scholar] [CrossRef] [Green Version]
  44. Bruss, V.; Ganem, D. The role of envelope proteins in hepatitis B virus assembly. Proc. Natl. Acad. Sci. USA 1991, 88, 1059–1063. [Google Scholar] [CrossRef] [PubMed]
  45. Zuccola, H.J.; Rozzelle, J.E.; Lemon, S.M.; Erickson, B.W.; Hogle, J.M. Structural basis of the oligomerization of hepatitis delta antigen. Structure 1998, 6, 821–830. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Hadziyannis, S.J.; Georgopoulou, U.; Psalidaki, E.; Budkowska, A. Pre-S1 and pre-S2 gene-encoded proteins in liver and serum in chronic hepatitis delta infection. J. Med. Virol. 1991, 34, 14–19. [Google Scholar] [CrossRef] [PubMed]
  47. Chang, J.; Nie, X.; Chang, H.E.; Han, Z.; Taylor, J. Transcription of Hepatitis Delta Virus RNA by RNA Polymerase II. J. Virol. 2008, 82, 1118–1127. [Google Scholar] [CrossRef] [Green Version]
  48. Chao, M.; Wang, T.-C.; Lin, C.-C.; Wang, R.Y.-L.; Lin, W.-B.; Lee, S.-E.; Cheng, Y.-Y.; Yeh, C.-T.; Iang, S.-B. Analyses of a whole-genome inter-clade recombination map of hepatitis delta virus suggest a host polymerase-driven and viral RNA structure-promoted template-switching mechanism for viral RNA recombination. Oncotarget 2017, 8, 60841–60859. [Google Scholar] [CrossRef] [Green Version]
  49. Ponzetto, A.; Hoyer, B.H.; Popper, H.; Engle, R.; Purcell, R.H.; Gerin, J.L. Titration of the Infectivity of Hepatitis D Virus in Chimpanzees. J. Infect. Dis. 1987, 155, 72–78. [Google Scholar] [CrossRef]
  50. Polakoff, S. Transmission from mother to infant of hepatitis B virus infection. Midwives Chron. 1983, 96, 4–5. [Google Scholar]
  51. Goudeau, A. Mother-to-infant transmission of hepatitis B virus. Towards the prevention of neonatal infection. Nouv. Presse Med. 1982, 11, 3051–3054. [Google Scholar]
  52. Rosenblum, L.; Darrow, W.; Witte, J.; Cohen, J.; French, J.; Gill, P.S.; Potterat, J.; Sikes, K.; Reich, R.; Hadler, S. Sexual Practices in the Transmission of Hepatitis B Virus and Prevalence of Hepatitis Delta Virus Infection in Female Prostitutes in the United States. JAMA 1992, 267, 2477–2481. [Google Scholar] [CrossRef]
  53. Taylor, J.M. Virus entry mediated by hepatitis B virus envelope proteins. World J. Gastroenterol. 2013, 19, 6730–6734. [Google Scholar] [CrossRef] [PubMed]
  54. Sureau, C. The Role of the HBV Envelope Proteins in the HDV Replication Cycle. Curr. Top. Microbiol. Immunol. 2006, 307, 113–131. [Google Scholar] [CrossRef] [PubMed]
  55. Yan, H.; Zhong, G.; Xu, G.; He, W.; Jing, Z.; Gao, Z.; Huang, Y.; Qi, Y.; Peng, B.; Wang, H.; et al. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. eLife 2012, 1, e00049. [Google Scholar] [CrossRef] [PubMed]
  56. Schulze, A.; Mills, K.; Weiss, T.S.; Urban, S. Hepatocyte polarization is essential for the productive entry of the hepatitis B virus. Hepatology 2011, 55, 373–383. [Google Scholar] [CrossRef] [PubMed]
  57. Gudima, S.; He, Y.; Meier, A.; Chang, J.; Chen, R.; Jarnik, M.; Nicolas, E.; Bruss, V.; Taylor, J. Assembly of Hepatitis Delta Virus: Particle Characterization, Including the Ability to Infect Primary Human Hepatocytes. J. Virol. 2007, 81, 3608–3617. [Google Scholar] [CrossRef] [Green Version]
  58. Longarela, O.L.; Schmidt, T.T.; Schöneweis, K.; Romeo, R.; Wedemeyer, H.; Urban, S.; Schulze, A. Proteoglycans Act as Cellular Hepatitis Delta Virus Attachment Receptors. PLoS ONE 2013, 8, e58340. [Google Scholar] [CrossRef] [Green Version]
  59. Engelke, M.; Mills, K.; Seitz, S.; Simon, P.; Gripon, P.; Schnölzer, M.; Urban, S. Characterization of a hepatitis B and hepatitis delta virus receptor binding site. Hepatology 2006, 43, 750–760. [Google Scholar] [CrossRef]
  60. Barrera, A.; Guerra, B.; Notvall, L.; Lanford, R.E. Mapping of the Hepatitis B Virus Pre-S1 Domain Involved in Receptor Recognition. J. Virol. 2005, 79, 9786–9798. [Google Scholar] [CrossRef] [Green Version]
  61. Murayama, A.; Yamada, N.; Osaki, Y.; Shiina, M.; Aly, H.H.; Iwamoto, M.; Tsukuda, S.; Watashi, K.; Matsuda, M.; Suzuki, R.; et al. N-Terminal PreS1 Sequence Regulates Efficient Infection of Cell-Culture–Generated Hepatitis B Virus. Hepatology 2020, 73, 520–532. [Google Scholar] [CrossRef]
  62. Huang, H.-C.; Chen, C.-C.; Chang, W.-C.; Tao, M.-H.; Huang, C. Entry of Hepatitis B Virus into Immortalized Human Primary Hepatocytes by Clathrin-Dependent Endocytosis. J. Virol. 2012, 86, 9443–9453. [Google Scholar] [CrossRef] [Green Version]
  63. Umetsu, T.; Inoue, J.; Kogure, T.; Kakazu, E.; Ninomiya, M.; Iwata, T.; Takai, S.; Nakamura, T.; Sano, A.; Shimosegawa, T. Inhibitory effect of silibinin on hepatitis B virus entry. Biochem. Biophys. Rep. 2018, 14, 20–25. [Google Scholar] [CrossRef] [PubMed]
  64. Herrscher, C.; Roingeard, P.; Blanchard, E. Hepatitis B Virus Entry into Cells. Cells 2020, 9, 1486. [Google Scholar] [CrossRef] [PubMed]
  65. Xia, Y.P.; Yeh, C.T.; Ou, J.H.; Lai, M.M. Characterization of nuclear targeting signal of hepatitis delta antigen: Nuclear transport as a protein complex. J. Virol. 1992, 66, 914–921. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  66. Cooper, A.; Shaul, Y. Clathrin-mediated Endocytosis and Lysosomal Cleavage of Hepatitis B Virus Capsid-like Core Particles. J. Biol. Chem. 2006, 281, 16563–16569. [Google Scholar] [CrossRef] [Green Version]
  67. Rabe, B.; Glebe, D.; Kann, M. Lipid-Mediated Introduction of Hepatitis B Virus Capsids into Nonsusceptible Cells Allows Highly Efficient Replication and Facilitates the Study of Early Infection Events. J. Virol. 2006, 80, 5465–5473. [Google Scholar] [CrossRef] [Green Version]
  68. Tuttleman, J.S.; Pourcel, C.; Summers, J. Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell 1986, 47, 451–460. [Google Scholar] [CrossRef]
  69. Guo, J.-T.; Guo, H. Metabolism and function of hepatitis B virus cccDNA: Implications for the development of cccDNA-targeting antiviral therapeutics. Antivir. Res. 2015, 122, 91–100. [Google Scholar] [CrossRef] [Green Version]
  70. Wang, K.-S.; Choo, Q.-L.; Weiner, A.J.; Ou, J.-H.; Najarian, R.C.; Thayer, R.M.; Mullenbach, G.T.; Denniston, K.J.; Gerin, J.L.; Houghton, M. Structure, sequence and expression of the hepatitis delta (δ) viral genome. Nature 1986, 323, 508–514. [Google Scholar] [CrossRef]
  71. Hsieh, S.Y.; Chao, M.; Coates, L.; Taylor, J. Hepatitis delta virus genome replication: A polyadenylated mRNA for delta antigen. J. Virol. 1990, 64, 3192–3198. [Google Scholar] [CrossRef] [Green Version]
  72. Chad, Y.-C.; Chang, M.-F.; Gust, I.; Lai, M.M. Sequence conservation and divergence of hepatitis virus RNA. Virology 1990, 178, 384–392. [Google Scholar] [CrossRef]
  73. Schieck, A.; Schulze, A.; Gähler, C.; Müller, T.; Haberkorn, U.; Alexandrov, A.; Urban, S.; Mier, W. Hepatitis B virus hepatotropism is mediated by specific receptor recognition in the liver and not restricted to susceptible hosts. Hepatology 2013, 58, 43–53. [Google Scholar] [CrossRef] [PubMed]
  74. Gripon, P.; Diot, C.; Thézé, N.; Fourel, I.; Loreal, O.; Brechot, C.; Guguen-Guillouzo, C. Hepatitis B virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide. J. Virol. 1988, 62, 4136–4143. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  75. Lucifora, J.; Durantel, D.; Testoni, B.; Hantz, O.; Levrero, M.; Zoulim, F. Control of hepatitis B virus replication by innate response of HepaRG cells. Hepatology 2009, 51, 63–72. [Google Scholar] [CrossRef] [PubMed]
  76. Luangsay, S.; Gruffaz, M.; Isorce, N.; Testoni, B.; Michelet, M.; Faure-Dupuy, S.; Maadadi, S.; Ait-Goughoulte, M.; Parent, R.; Rivoire, M.; et al. Early inhibition of hepatocyte innate responses by hepatitis B virus. J. Hepatol. 2015, 63, 1314–1322. [Google Scholar] [CrossRef]
  77. Liu, Y.; Li, J.; Chen, J.; Li, Y.; Wang, W.; Du, X.; Song, W.; Zhang, W.; Lin, L.; Yuan, Z. Hepatitis B Virus Polymerase Disrupts K63-Linked Ubiquitination of STING To Block Innate Cytosolic DNA-Sensing Pathways. J. Virol. 2015, 89, 2287–2300. [Google Scholar] [CrossRef] [Green Version]
  78. Verrier, E.R.; Colpitts, C.C.; Schuster, C.; Zeisel, M.B.; Baumert, T.F. Cell Culture Models for the Investigation of Hepatitis B and D Virus Infection. Viruses 2016, 8, 261. [Google Scholar] [CrossRef]
  79. Alfaiate, D.; Lucifora, J.; Abeywickrama-Samarakoon, N.; Michelet, M.; Testoni, B.; Cortay, J.-C.; Sureau, C.; Zoulim, F.; Dény, P.; Durantel, D. HDV RNA replication is associated with HBV repression and interferon-stimulated genes induction in super-infected hepatocytes. Antivir. Res. 2016, 136, 19–31. [Google Scholar] [CrossRef]
  80. Mabit, H.; Dubanchet, S.; Capel, F.; Dauguet, C.; Petit, M.-A. In vitro infection of human hepatoma cells (HepG2) with hepatitis B virus (HBV): Spontaneous selection of a stable HBV surface antigen-producing HepG2 cell line containing integrated HBV DNA sequences. J. Gen. Virol. 1994, 75 Pt 10, 2681–2689. [Google Scholar] [CrossRef]
  81. König, A.; Döring, B.; Mohr, C.; Geipel, A.; Geyer, J.; Glebe, D. Kinetics of the bile acid transporter and hepatitis B virus receptor Na+/taurocholate cotransporting polypeptide (NTCP) in hepatocytes. J. Hepatol. 2014, 61, 867–875. [Google Scholar] [CrossRef] [Green Version]
  82. Levy, G.; Bomze, D.; Heinz, S.; Ramachandran, S.D.; Noerenberg, A.; Cohen, M.M.; Shibolet, O.; Sklan, E.H.; Braspenning, J.; Nahmias, Y. Long-term culture and expansion of primary human hepatocytes. Nat. Biotechnol. 2015, 33, 1264–1271. [Google Scholar] [CrossRef]
  83. Gural, N.; Mancio-Silva, L.; He, J.; Bhatia, S.N. Engineered Livers for Infectious Diseases. Cell. Mol. Gastroenterol. Hepatol. 2017, 5, 131–144. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  84. Kidambi, S.; Yarmush, R.S.; Novik, E.; Chao, P.; Yarmush, M.L.; Nahmias, Y. Oxygen-mediated enhancement of primary hepatocyte metabolism, functional polarization, gene expression, and drug clearance. Proc. Natl. Acad. Sci. USA 2009, 106, 15714–15719. [Google Scholar] [CrossRef] [PubMed]
  85. Yoneda, M.; Hyun, J.; Jakubski, S.; Saito, S.; Nakajima, A.; Schiff, E.R.; Thomas, E. Hepatitis B Virus and DNA Stimulation Trigger a Rapid Innate Immune Response through NF-κB. J. Immunol. 2016, 197, 630–643. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  86. Ortega-Prieto, A.M.; Skelton, J.K.; Wai, S.N.; Large, E.; Lussignol, M.; Vizcay-Barrena, G.; Hughes, D.; Fleck, R.A.; Thursz, M.; Catanese, M.T.; et al. 3D microfluidic liver cultures as a physiological preclinical tool for hepatitis B virus infection. Nat. Commun. 2018, 9, 682. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  87. Zhang, Z.; Trippler, M.; Real, C.I.; Werner, M.; Luo, X.; Schefczyk, S.; Kemper, T.; Anastasiou, O.E.; Ladiges, Y.; Treckmann, J.; et al. Hepatitis B Virus Particles Activate Toll-Like Receptor 2 Signaling Initially Upon Infection of Primary Human Hepatocytes. Hepatology 2020, 72, 829–844. [Google Scholar] [CrossRef] [Green Version]
  88. Zeisel, M.B.; Lucifora, J.; Mason, W.S.; Sureau, C.; Beck, J.; Levrero, M.; Kann, M.; Knolle, P.A.; Benkirane, M.; Durantel, D.; et al. Towards an HBV cure: State-of-the-art and unresolved questions—Report of the ANRS workshop on HBV cure. Gut 2015, 64, 1314–1326. [Google Scholar] [CrossRef]
  89. Bhogal, R.H.; Hodson, J.; Bartlett, D.C.; Weston, C.J.; Curbishley, S.M.; Haughton, E.; Williams, K.T.; Reynolds, G.M.; Newsome, P.N.; Adams, D.H.; et al. Isolation of Primary Human Hepatocytes from Normal and Diseased Liver Tissue: A One Hundred Liver Experience. PLoS ONE 2011, 6, e18222. [Google Scholar] [CrossRef] [Green Version]
  90. Zhou, M.; Zhao, F.; Li, J.; Cheng, Z.; Tian, X.; Zhi, X.; Huang, Y.; Hu, K. Long-term maintenance of human fetal hepatocytes and prolonged susceptibility to HBV infection by co-culture with non-parenchymal cells. J. Virol. Methods 2013, 195, 185–193. [Google Scholar] [CrossRef]
  91. Godoy, P.; Hewitt, N.J.; Albrecht, U.; Andersen, M.E.; Ansari, N.; Bhattacharya, S.; Bode, J.G.; Bolleyn, J.; Borner, C.; Böttger, J.; et al. Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch. Toxicol. 2013, 87, 1315–1530. [Google Scholar] [CrossRef] [Green Version]
  92. Shlomai, A.; Schwartz, R.E.; Ramanan, V.; Bhatta, A.; de Jong, Y.P.; Bhatia, S.N.; Rice, C.M. Modeling host interactions with hepatitis B virus using primary and induced pluripotent stem cell-derived hepatocellular systems. Proc. Natl. Acad. Sci. USA 2014, 111, 12193–12198. [Google Scholar] [CrossRef]
  93. Winer, B.Y.; Huang, T.S.; Pludwinski, E.; Heller, B.; Wojcik, F.; Lipkowitz, G.E.; Parekh, A.; Cho, C.; Shrirao, A.; Muir, T.W.; et al. Long-term hepatitis B infection in a scalable hepatic co-culture system. Nat. Commun. 2017, 8, 125. [Google Scholar] [CrossRef] [Green Version]
  94. Winer, B.Y.; Gaska, J.M.; Lipkowitz, G.; Bram, Y.; Parekh, A.; Parsons, L.; Leach, R.; Jindal, R.; Cho, C.H.; Shrirao, A.; et al. Analysis of Host Responses to Hepatitis B and Delta Viral Infections in a Micro-scalable Hepatic Co-culture System. Hepatology 2019, 71, 14–30. [Google Scholar] [CrossRef] [PubMed]
  95. Zhang, Z.; Filzmayer, C.; Ni, Y.; Sültmann, H.; Mutz, P.; Hiet, M.-S.; Vondran, F.W.; Bartenschlager, R.; Urban, S. Hepatitis D virus replication is sensed by MDA5 and induces IFN-β/λ responses in hepatocytes. J. Hepatol. 2018, 69, 25–35. [Google Scholar] [CrossRef]
  96. Gripon, P.; Rumin, S.; Urban, S.; Le Seyec, J.; Glaise, D.; Cannie, I.; Guyomard, C.; Lucas, J.; Trepo, C.; Guguen-Guillouzo, C. Infection of a human hepatoma cell line by hepatitis B virus. Proc. Natl. Acad. Sci. USA 2002, 99, 15655–15660. [Google Scholar] [CrossRef]
  97. Aninat, C.; Piton, A.; Glaise, D.; Le Charpentier, T.; Langouët, S.; Morel, F.; Guguen-Guillouzo, C.; Guillouzo, A. Expression of cytochromes P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells. Drug Metab. Dispos. 2005, 34, 75–83. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  98. Guillouzo, A.; Corlu, A.; Aninat, C.; Glaise, D.; Morel, F.; Guguen-Guillouzo, C. The human hepatoma HepaRG cells: A highly differentiated model for studies of liver metabolism and toxicity of xenobiotics. Chem. Interact. 2007, 168, 66–73. [Google Scholar] [CrossRef]
  99. Turpeinen, M.; Tolonen, A.; Chesne, C.; Guillouzo, A.; Uusitalo, J.; Pelkonen, O. Functional expression, inhibition and induction of CYP enzymes in HepaRG cells. Toxicol. Vitr. 2009, 23, 748–753. [Google Scholar] [CrossRef] [PubMed]
  100. Kanebratt, K.P.; Andersson, T.B. Evaluation of HepaRG Cells as an in Vitro Model for Human Drug Metabolism Studies. Drug Metab. Dispos. 2008, 36, 1444–1452. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  101. Hantz, O.; Parent, R.; Durantel, D.; Gripon, P.; Guguen-Guillouzo, C.; Zoulim, F. Persistence of the hepatitis B virus covalently closed circular DNA in HepaRG human hepatocyte-like cells. J. Gen. Virol. 2009, 90, 127–135. [Google Scholar] [CrossRef]
  102. Jennen, D.G.J.; Magkoufopoulou, C.; Ketelslegers, H.B.; van Herwijnen, M.H.M.; Kleinjans, J.C.S.; van Delft, J.H.M. Comparison of HepG2 and HepaRG by Whole-Genome Gene Expression Analysis for the Purpose of Chemical Hazard Identification. Toxicol. Sci. 2010, 115, 66–79. [Google Scholar] [CrossRef] [Green Version]
  103. Gerets, H.H.J.; Tilmant, K.; Gerin, B.; Chanteux, H.; Depelchin, B.O.; Dhalluin, S.; Atienzar, F.A. Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins. Cell Biol. Toxicol. 2012, 28, 69–87. [Google Scholar] [CrossRef] [Green Version]
  104. Macovei, A.; Radulescu, C.; Lazar, C.; Petrescu, S.; Durantel, D.; Dwek, R.A.; Zitzmann, N.; Nichita, N.B. Hepatitis B Virus Requires Intact Caveolin-1 Function for Productive Infection in HepaRG Cells. J. Virol. 2010, 84, 243–253. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  105. Mutz, P.; Metz, P.; Lempp, F.A.; Bender, S.; Qu, B.; Schöneweis, K.; Seitz, S.; Tu, T.; Restuccia, A.; Frankish, J.; et al. HBV Bypasses the Innate Immune Response and Does Not Protect HCV From Antiviral Activity of Interferon. Gastroenterology 2018, 154, 1791–1804.e22. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  106. Andersson, T.B.; Kanebratt, K.P.; Kenna, J.G. The HepaRG cell line: A unique in vitro tool for understanding drug metabolism and toxicology in human. Expert Opin. Drug Metab. Toxicol. 2012, 8, 909–920. [Google Scholar] [CrossRef] [PubMed]
  107. Phillips, S.; Chokshi, S.; Chatterji, U.; Riva, A.; Bobardt, M.; Williams, R.; Gallay, P.; Naoumov, N.V. Alisporivir Inhibition of Hepatocyte Cyclophilins Reduces HBV Replication and Hepatitis B Surface Antigen Production. Gastroenterology 2015, 148, 403–414.e7. [Google Scholar] [CrossRef] [PubMed]
  108. Nunn, A.D.G.; Scopigno, T.; Pediconi, N.; Levrero, M.; Hagman, H.; Kiskis, J.; Enejder, A. The histone deacetylase inhibiting drug Entinostat induces lipid accumulation in differentiated HepaRG cells. Sci. Rep. 2016, 6, 28025. [Google Scholar] [CrossRef] [PubMed]
  109. Mayati, A.; Moreau, A.; Le Vée, M.; Bruyère, A.; Jouan, E.; Denizot, C.; Parmentier, Y.; Fardel, O. Functional polarization of human hepatoma HepaRG cells in response to forskolin. Sci. Rep. 2018, 8, 16115. [Google Scholar] [CrossRef] [Green Version]
  110. Xiang, C.; Du, Y.; Meng, G.; Yi, L.S.; Sun, S.; Song, N.; Zhang, X.; Xiao, Y.; Wang, J.; Yi, Z.; et al. Long-term functional maintenance of primary human hepatocytes in vitro. Science 2019, 364, 399–402. [Google Scholar] [CrossRef]
  111. Lucifora, J.; Michelet, M.; Salvetti, A.; Durantel, D. Fast Differentiation of HepaRG Cells Allowing Hepatitis B and Delta Virus Infections. Cells 2020, 9, 2288. [Google Scholar] [CrossRef]
  112. Nakabayashi, H.; Taketa, K.; Miyano, K.; Yamane, T.; Sato, J. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res. 1982, 42, 3858–3863. [Google Scholar]
  113. Chiu, J.-H.; Hu, C.-P.; Lui, W.-Y.; Lo, S.J.; Chang, C. The formation of bile canaliculi in human hepatoma cell lines. Hepatology 1990, 11, 834–842. [Google Scholar] [CrossRef] [PubMed]
  114. Meier, A.; Mehrle, S.; Weiss, T.S.; Mier, W.; Urban, S. Myristoylated PreS1-domain of the hepatitis B virus L-protein mediates specific binding to differentiated hepatocytes. Hepatology 2012, 58, 31–42. [Google Scholar] [CrossRef]
  115. Verrier, E.R.; Colpitts, C.C.; Bach, C.; Heydmann, L.; Weiss, A.; Renaud, M.; Durand, S.C.; Habersetzer, F.; Durantel, D.; Abou-Jaoudé, G.; et al. A targeted functional RNA interference screen uncovers glypican 5 as an entry factor for hepatitis B and D viruses. Hepatology 2016, 63, 35–48. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  116. Ni, Y.; Zhang, Z.; Engelskircher, L.; Verch, G.; Tu, T.; Lempp, F.A.; Urban, S. Generation and characterization of a stable cell line persistently replicating and secreting the human hepatitis delta virus. Sci. Rep. 2019, 9, 10021. [Google Scholar] [CrossRef] [Green Version]
  117. Le, C.; Sirajee, R.; Steenbergen, R.; Joyce, M.A.; Addison, W.R.; Tyrrell, D.L. In Vitro Infection with Hepatitis B Virus Using Differentiated Human Serum Culture of Huh7.5-NTCP Cells without Requiring Dimethyl Sulfoxide. Viruses 2021, 13, 97. [Google Scholar] [CrossRef] [PubMed]
  118. Vondráček, J.; Souček, K.; Sheard, M.A.; Chramostová, K.; Andrysík, Z.; Hofmanová, J.; Kozubík, A. Dimethyl sulfoxide potentiates death receptor-mediated apoptosis in the human myeloid leukemia U937 cell line through enhancement of mitochondrial membrane depolarization. Leuk. Res. 2006, 30, 81–89. [Google Scholar] [CrossRef]
  119. Steenbergen, R.H.; Joyce, M.A.; Thomas, B.S.; Jones, D.; Law, J.; Russell, R.; Houghton, M.; Tyrrell, D.L. Human serum leads to differentiation of human hepatoma cells, restoration of very-low-density lipoprotein secretion, and a 1000-fold increase in HCV Japanese fulminant hepatitis type 1 titers. Hepatology 2013, 58, 1907–1917. [Google Scholar] [CrossRef]
  120. Galvao, J.; Davis, B.; Tilley, M.; Normando, E.; Duchen, M.R.; Cordeiro, M.F. Unexpected low-dose toxicity of the universal solvent DMSO. FASEB J. 2013, 28, 1317–1330. [Google Scholar] [CrossRef]
  121. Ni, Y.; Lempp, F.A.; Mehrle, S.; Nkongolo, S.; Kaufman, C.; Fälth, M.; Stindt, J.; Königer, C.; Nassal, M.; Kubitz, R.; et al. Hepatitis B and D Viruses Exploit Sodium Taurocholate Co-transporting Polypeptide for Species-Specific Entry into Hepatocytes. Gastroenterology 2014, 146, 1070–1083.e6. [Google Scholar] [CrossRef]
  122. Altstetter, S.M.; Quitt, O.; Pinci, F.; Hornung, V.; Lucko, A.M.; Wisskirchen, K.; Jung, S.; Protzer, U. Hepatitis-D Virus Infection Is Not Impaired by Innate Immunity but Increases Cytotoxic T-Cell Activity. Cells 2021, 10, 3253. [Google Scholar] [CrossRef]
  123. Pugnale, P.; Pazienza, V.; Guilloux, K.; Negro, F. Hepatitis delta virus inhibits alpha interferon signaling. Hepatology 2009, 49, 398–406. [Google Scholar] [CrossRef] [PubMed]
  124. Sassa, S.; Sugita, O.; Galbraith, R.A.; Kappas, A. Drug metabolism by the human hepatoma cell, Hep G2. Biochem. Biophys. Res. Commun. 1987, 143, 52–57. [Google Scholar] [CrossRef] [PubMed]
  125. Olsavsky, K.M.; Page, J.L.; Johnson, M.C.; Zarbl, H.; Strom, S.C.; Omiecinski, C.J. Gene expression profiling and differentiation assessment in primary human hepatocyte cultures, established hepatoma cell lines, and human liver tissues. Toxicol. Appl. Pharmacol. 2007, 222, 42–56. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  126. Westerink, W.M.; Schoonen, W.G. Cytochrome P450 enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells. Toxicol. Vitr. 2007, 21, 1581–1591. [Google Scholar] [CrossRef]
  127. Sells, M.A.; Chen, M.L.; Acs, G. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA. Proc. Natl. Acad. Sci. USA 1987, 84, 1005–1009. [Google Scholar] [CrossRef]
  128. Ladner, S.K.; Otto, M.J.; Barker, C.S.; Zaifert, K.; Wang, G.H.; Guo, J.T.; Seeger, C.; King, R.W. Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: A novel system for screening potential inhibitors of HBV replication. Antimicrob. Agents Chemother. 1997, 41, 1715–1720. [Google Scholar] [CrossRef] [Green Version]
  129. Ding, X.R.; Yang, J.; Sun, D.C.; Lou, S.K.; Wang, S.Q. Whole genome expression profiling of hepatitis B virus-transfected cell line reveals the potential targets of anti-HBV drugs. Pharmacogenomics J. 2007, 8, 61–70. [Google Scholar] [CrossRef] [Green Version]
  130. Li, G.-Q.; Xu, W.-Z.; Wang, J.-X.; Deng, W.-W.; Li, D.; Gu, H.-X. Combination of small interfering RNA and lamivudine on inhibition of human B virus replication in HepG2.2.15 cells. World J. Gastroenterol. 2007, 13, 2324–2327. [Google Scholar] [CrossRef]
  131. Xin, X.-M.; Li, G.-Q.; Guan, X.-R.; Li, D.; Xu, W.-Z.; Jin, Y.-Y.; Gu, H.-X. Combination therapy of siRNAs mediates greater suppression on hepatitis B virus cccDNA in HepG2.2.15 cell. Hepato-Gastroenterology 2009, 55, 2178–2183. [Google Scholar]
  132. Michailidis, E.; Pabon, J.; Xiang, K.; Park, P.; Ramanan, V.; Hoffmann, H.-H.; Schneider, W.M.; Bhatia, S.N.; de Jong, Y.P.; Shlomai, A.; et al. A robust cell culture system supporting the complete life cycle of hepatitis B virus. Sci. Rep. 2017, 7, 16616. [Google Scholar] [CrossRef] [Green Version]
  133. Li, X.; Xu, Z.; Mitra, B.; Wang, M.; Guo, H.; Feng, Z. Elevated NTCP expression by an iPSC-derived human hepatocyte maintenance medium enhances HBV infection in NTCP-reconstituted HepG2 cells. Cell Biosci. 2021, 11, 123. [Google Scholar] [CrossRef] [PubMed]
  134. Li, W.; Urban, S. Entry of hepatitis B and hepatitis D virus into hepatocytes: Basic insights and clinical implications. J. Hepatol. 2016, 64, S32–S40. [Google Scholar] [CrossRef] [PubMed]
  135. Harris, A.J.; Dial, S.L.; Casciano, D.A. Comparison of basal gene expression profiles and effects of hepatocarcinogens on gene expression in cultured primary human hepatocytes and HepG2 cells. Mutat. Res. Mol. Mech. Mutagen. 2004, 549, 79–99. [Google Scholar] [CrossRef]
  136. Liguori, M.J.; Blomme, E.A.; Waring, J.F. Trovafloxacin-Induced Gene Expression Changes in Liver-Derived in Vitro Systems: Comparison of Primary Human Hepatocytes to HepG2 Cells. Drug Metab. Dispos. 2007, 36, 223–233. [Google Scholar] [CrossRef] [PubMed]
  137. Coulouarn, C.; Factor, V.M.; Andersen, J.B.; Durkin, M.E.; Thorgeirsson, S.S. Loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties. Oncogene 2009, 28, 3526–3536. [Google Scholar] [CrossRef] [Green Version]
  138. Song, Y.M.; Song, S.-O.; Jung, Y.-K.; Kang, E.-S.; Cha, B.-S.; Lee, H.C.; Lee, B.-W. Dimethyl sulfoxide reduces hepatocellular lipid accumulation through autophagy induction. Autophagy 2012, 8, 1085–1097. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  139. Pal, R.; Mamidi, M.K.; Das, A.K.; Bhonde, R. Diverse effects of dimethyl sulfoxide (DMSO) on the differentiation potential of human embryonic stem cells. Arch. Toxicol. 2011, 86, 651–661. [Google Scholar] [CrossRef]
  140. Verheijen, M.; Lienhard, M.; Schrooders, Y.; Clayton, O.; Nudischer, R.; Boerno, S.; Timmermann, B.; Selevsek, N.; Schlapbach, R.; Gmuender, H.; et al. DMSO induces drastic changes in human cellular processes and epigenetic landscape in vitro. Sci. Rep. 2019, 9, 4641. [Google Scholar] [CrossRef] [Green Version]
  141. Gripon, P.; Diot, C.; Guguen Guillouzo, C. Reproducible High Level Infection of Cultured Adult Human Hepatocytes by Hepatitis B Virus: Effect of Polyethylene Glycol on Adsorption and Penetration. Virology 1993, 192, 534–540. [Google Scholar] [CrossRef]
  142. Schulze, A.; Gripon, P.; Urban, S. Hepatitis B virus infection initiates with a large surface protein-dependent binding to heparan sulfate proteoglycans. Hepatology 2007, 46, 1759–1768. [Google Scholar] [CrossRef]
  143. Urban, S.; Bartenschlager, R.; Kubitz, R.; Zoulim, F. Strategies to Inhibit Entry of HBV and HDV Into Hepatocytes. Gastroenterology 2014, 147, 48–64. [Google Scholar] [CrossRef] [PubMed]
  144. König, A.; Yang, J.; Jo, E.; Park, K.H.P.; Kim, H.; Than, T.T.; Song, X.; Qi, X.; Dai, X.; Park, S.; et al. Efficient long-term amplification of hepatitis B virus isolates after infection of slow proliferating HepG2-NTCP cells. J. Hepatol. 2019, 71, 289–300. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  145. Zahoor, M.A.; Kuipery, A.; Mosa, A.I.; Gehring, A.J.; Feld, J.J. HepG2-NTCP Subclones Exhibiting High Susceptibility to Hepatitis B Virus Infection. Viruses 2022, 14, 1800. [Google Scholar] [CrossRef]
  146. Song, Y.; Shou, S.; Guo, H.; Gao, Z.; Liu, N.; Yang, Y.; Wang, F.; Deng, Q.; Liu, J.; Xie, Y. Establishment and characterization of a new cell culture system for hepatitis B virus replication and infection. Virol. Sin. 2022, 37, 558–568. [Google Scholar] [CrossRef] [PubMed]
  147. Boess, F.; Kamber, M.; Romer, S.; Gasser, R.; Muller, D.; Albertini, S.; Suter, L. Gene Expression in Two Hepatic Cell Lines, Cultured Primary Hepatocytes, and Liver Slices Compared to the in Vivo Liver Gene Expression in Rats: Possible Implications for Toxicogenomics Use of in Vitro Systems. Toxicol. Sci. 2003, 73, 386–402. [Google Scholar] [CrossRef] [Green Version]
  148. Rodríguez-Antona, C.; Donato, M.T.; Boobis, A.; Edwards, R.J.; Watts, P.S.; Castell, J.V.; Gómez-Lechón, M.-J. Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: Molecular mechanisms that determine lower expression in cultured cells. Xenobiotica 2002, 32, 505–520. [Google Scholar] [CrossRef] [PubMed]
  149. Nelson, L.J.; Walker, S.W.; Hayes, P.C.; Plevris, J.N. Low-Shear Modelled Microgravity Environment Maintains Morphology and Differentiated Functionality of Primary Porcine Hepatocyte Cultures. Cells Tissues Organs 2010, 192, 125–140. [Google Scholar] [CrossRef] [Green Version]
  150. Richert, L.; Binda, D.; Hamilton, G.; Viollon-Abadie, C.; Alexandre, E.; Bigot-Lasserre, D.; Bars, R.; Coassolo, P.; LeCluyse, E. Evaluation of the effect of culture configuration on morphology, survival time, antioxidant status and metabolic capacities of cultured rat hepatocytes. Toxicol. Vitr. 2002, 16, 89–99. [Google Scholar] [CrossRef]
  151. Schyschka, L.; Sánchez, J.J.M.; Wang, Z.; Burkhardt, B.; Müller-Vieira, U.; Zeilinger, K.; Bachmann, A.; Nadalin, S.; Damm, G.; Nussler, A.K. Hepatic 3D cultures but not 2D cultures preserve specific transporter activity for acetaminophen-induced hepatotoxicity. Arch. Toxicol. 2013, 87, 1581–1593. [Google Scholar] [CrossRef]
  152. Shulman, M.; Nahmias, Y. Long-Term Culture and Coculture of Primary Rat and Human Hepatocytes. In Epithelial Cell Culture Protocols; Humana Press: Totowa, NJ, USA, 2012; Volume 945, pp. 287–302. [Google Scholar] [CrossRef] [Green Version]
  153. Kukla, D.A.; Crampton, A.L.; Wood, D.K.; Khetani, S.R. Microscale Collagen and Fibroblast Interactions Enhance Primary Human Hepatocyte Functions in Three-Dimensional Models. Gene Expr. 2020, 20, 1–18. [Google Scholar] [CrossRef]
  154. Vinken, M.; Decrock, E.; Doktorova, T.; Ramboer, E.; De Vuyst, E.; Vanhaecke, T.; Leybaert, L.; Rogiers, V. Characterization of spontaneous cell death in monolayer cultures of primary hepatocytes. Arch. Toxicol. 2011, 85, 1589–1596. [Google Scholar] [CrossRef] [PubMed]
  155. Ullah, I.; Kim, Y.; Lim, M.; Oh, K.B.; Hwang, S.; Shin, Y.; Kim, Y.; Im, G.-S.; Hur, T.-Y.; A Ock, S. In vitro 3-D culture demonstrates incompetence in improving maintenance ability of primary hepatocytes. Anim. Cells Syst. 2017, 21, 332–340. [Google Scholar] [CrossRef]
  156. Sainz, B., Jr.; TenCate, V.; Uprichard, S.L. Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection. Virol. J. 2009, 6, 103. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  157. Rajalakshmy, A.; Malathi, J.; Madhavan, H.; Samuel, J. Mebiolgel, a thermoreversible polymer as a scaffold for three dimensional culture of Huh7 cell line with improved hepatocyte differentiation marker expression and HCV replication. Indian J. Med. Microbiol. 2015, 33, 554–559. [Google Scholar] [CrossRef] [PubMed]
  158. Allweiss, L.; Dandri, M. Experimental in vitro and in vivo models for the study of human hepatitis B virus infection. J. Hepatol. 2016, 64, S17–S31. [Google Scholar] [CrossRef]
  159. Nassal, M. HBV cccDNA: Viral persistence reservoir and key obstacle for a cure of chronic hepatitis B. Gut 2015, 64, 1972–1984. [Google Scholar] [CrossRef] [Green Version]
Life 13 01527 g001 550
Figure 1. Structure of HBV and HDV virions. Both virions share HBV surface proteins, S-, M-, and L-HBsAg on their envelope. HBV contains a double-stranded DNA genome (or relaxed circular DNA, rcDNA) and the viral DNA polymerase, while HDV has a negative sense single-stranded circular RNA associated with the small delta antigen (S-HDAg) and large delta antigen (L-HDAg).
Figure 1. Structure of HBV and HDV virions. Both virions share HBV surface proteins, S-, M-, and L-HBsAg on their envelope. HBV contains a double-stranded DNA genome (or relaxed circular DNA, rcDNA) and the viral DNA polymerase, while HDV has a negative sense single-stranded circular RNA associated with the small delta antigen (S-HDAg) and large delta antigen (L-HDAg).
Life 13 01527 g001
Table
Table 1. Advantages and limitations of the cell culture system supporting HBV and/or HDV infection.
Table 1. Advantages and limitations of the cell culture system supporting HBV and/or HDV infection.
CellsAdvantagesLimitations
PHH• Supports the complete life cycle of HBV and HDV infections• Limited availability of high-quality donors and lifespan
• Contains various hepatocyte-specific host factors • Variable susceptibility to HBV/HDV infection
• Exhibits a fully functional innate immune system• Loss of their differentiation functions after plating
• Difficult to manage in cultural conditions
HepaRG• Contains hepatic functions• Low infection efficiency
• Expresses transcripts of various nuclear receptors• Requires differentiation
• Limited cell-to-cell spreading
Huh7-NTCP• Better infection efficiencies for HDV infection • Partially mimic normal hepatocytes due to poor polarization
• No detectable amounts of the receptor
HepG2-NTCP• Readily available• Partially mimic normal hepatocytes
• High reproducibility• Low infection efficiency/limited viral replication
• Robust viral infection• Require extra usage of PEG and DMSO
HepG2-NTCP sec+• Supports the complete HBV life cycle• Require high viral titer inoculum
• Long-term viral spread• Require PEG to increase viral infectivity
The 3D culture• Maintains an intact morphology• Does not fully maintain a natural hepatic environment and liver-specific functions
• No PEG or DMSO needed
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Lee, G.S.; Purdy, M.A.; Choi, Y. Cell Culture Systems for Studying Hepatitis B and Hepatitis D Virus Infections. Life 2023, 13, 1527. https://doi.org/10.3390/life13071527

AMA Style

Lee GS, Purdy MA, Choi Y. Cell Culture Systems for Studying Hepatitis B and Hepatitis D Virus Infections. Life. 2023; 13(7):1527. https://doi.org/10.3390/life13071527

Chicago/Turabian Style

Lee, Grace Sanghee, Michael A. Purdy, and Youkyung Choi. 2023. "Cell Culture Systems for Studying Hepatitis B and Hepatitis D Virus Infections" Life 13, no. 7: 1527. https://doi.org/10.3390/life13071527

APA Style

Lee, G. S., Purdy, M. A., & Choi, Y. (2023). Cell Culture Systems for Studying Hepatitis B and Hepatitis D Virus Infections. Life, 13(7), 1527. https://doi.org/10.3390/life13071527

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop