Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 11, pp. 1504-1520 © Pleiades Publishing, Ltd., 2025.

Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 11, pp. 1601-1620.

1504

REVIEW

In vivo and in vitro Models of Hepatitis B Virus Infection

Yuliya V. Kolyako

1,a

*, Alla S. Zhitkevich

, Daria V. Avdoshina

, Daria Y. Tanygina

Vasiliy D. Apolokhov

, Tatiana V. Gorodnicheva

1,2

, Dmitry S. Kostyushev

3,4,5

Ekaterina O. Bayurova

, and Ilya V. Gordeychuk

1,3

Chumakov Federal Scientific Center for Research and Development

of Immune-and-Biological Products of Russian Academy of Sciences (Institute of Poliomyelitis),

108819 Moscow, Russia

Pirogov Russian National Research Medical University, 117997 Moscow, Russia

Sechenov First Moscow State Medical University

of the Ministry of Health of the Russian Federation (Sechenov University), 117418 Moscow, Russia

Faculty of bioengineering and bioinformatics, Lomonosov Moscow State University, 119192 Moscow, Russia

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia

e-mail: kolyako_jv@chumakovs.su

Received July 23, 2025

Revised September 24, 2025

Accepted September 25, 2025

Abstract— Hepatitis B virus (Orthohepadnavirus hominoidei, HBV) is a hepatotropic virus from the Hepad-

naviridae family and the causative agent of both acute and chronic hepatitis B (CHB). The possible outcomes

of CHB include liver cirrhosis and hepatocellular carcinoma (HCC) that pose a significant burden on the

healthcare systems worldwide. In the nuclei of infected hepatocytes of patients with CHB, the HBV genome

persists as a pool of covalently closed circular DNA (cccDNA) molecules. Current therapeutic strategies cannot

directly target cccDNA. Instead, the available treatments focus on long-term suppression of viral replication

and require lifelong administration. Development and evaluation of novel antiviral agents capable of achiev-

ing complete HBV eradication require relevant in  vivo and in  vitro models of HBV infection. Among the

available animal models, the following categories are distinguished: (i)  animals naturally susceptible to HBV;

(ii)  surrogate models using animal species susceptible to the related hepadnaviruses; (iii)  non-susceptible ani-

mals receiving HBV genome via recombinant viral vectors; (iv)  models utilizing human hepatocyte xenografts.

Among the available in vitro models, primary human and northern treeshrew (Tupaia belangeri) hepato-

cytes fully support the HBV replication cycle, but they rapidly lose susceptibility to the virus in cell culture.

In turn, unmodified human hepatoma cell lines are not susceptible to HBV but can support viral replication

after transfection with the viral genome. This review discusses key characteristics, advantages, limitations,

and areas of application of the currently available invivo and in vitro models of HBV infection.

DOI: 10.1134/S0006297925602230

Keywords: hepatitis B virus, hepatitis B virus infection, chronic hepatitis B, viral infection models, laboratory

animals

* To whom correspondence should be addressed.

INTRODUCTION

According to the WHO estimates, approximately

1.2 million new cases of hepatitis  B virus (HBV) infec-

tion are recorded annually and more than 250 mil-

lion people are living with chronic hepatitis  B (CHB)

(https://www.who.int/news-room/fact-sheets/detail/

hepatitis-b, accessed 10.07.25). The HBV genome is rep-

resented by relaxed circular partially double-stranded

DNA (rcDNA) (~3200 base pairs) that contains four

overlapping open reading frames (ORF): S, pre-C/C,

P and X. ORF S includes pre-S1, pre-S2, and S re-

gions, which encode three isoforms of the surface

protein (HBsAg): small (SHBsAg), middle (MHBsAg)

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BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

and large (LHBsAg), all sharing a common C-termi-

nus but differing in N-terminal extensions and dif-

ferent glycosylation levels. ORF pre-C/C encodes pre-

core protein (HBeAg) and core protein (HBcAg). ORF

P encodes polymerase with reverse transcriptase and

RNase H activities. ORF X encodes a key transcription

activation factor (HBxAg) [1]. HBV hepatotropism is

determined by three factors: (i)  the unique receptor

expressed on hepatocyte surface, (ii)  HBV capsid nu-

clear import, and (iii)  hepatocyte-specific factors for

viral transcription. HBV infection begins with low-af-

finity binding of the virus to heparan sulfates pro-

teoglycans on the cell surface, followed by specific

interaction between the pre-S1 domain of LHBsAg

and its receptor – sodium-taurocholate cotransport-

ing polypeptide (NTCP). Next, the HBV/NTCP complex

is internalized into cells by interacting with the epi-

dermal growth factor receptor (EGFR) via clathrin-

dependent endocytosis. HBV enters the endolysosomal

compartment, where the rcDNA-containing capsid is

released into the cytosol. Import of the viral genome

into the nucleus is ensured by the interaction of the

nuclear localization signal in the terminal region of

HBcAg on the surface of the viral capsid with the

importin α1 [2]. In the nucleus, the HBV genome

is released from a capsid followed by conversion

of rcDNA into covalently closed circular DNA (cccDNA)

in a multistep process mediated by cellular enzymes.

The host RNA polymerase II mediates cccDNA tran-

scription into pregenomic RNA (pgRNA), which serves

as a template for reverse transcription of the(−) strand

of rcDNA [3]. The hepatocyte-specific factors such

as hepatocyte nuclear factors (HNF) HNF1α, HNF3β,

HNF4α, HNF6, and factor C/EBP, play a crucial role in

the HBV replication cycle [4]. The rcDNA is released

from cells within denovo virions or re-imported into

the nucleus, thereby ensuring maintenance of the in-

tranuclear pool of cccDNA [5].

HBV causes both acute and chronic infection,

transmitted from mother to child or via bloodborne

transmission. Incubation period of the infection varies

from 30 to 180 days. In most cases, acute infection in

adults is self-limiting and results in virus elimination.

However, in rare cases, fulminant hepatitis develops

followed by liver failure and patient death. CHB is

characterized by a long-term inflammatory process in

the liver eventually leading to the development of liver

cirrhosis and hepatocellular carcinoma (HCC). The risk

of developing CHB in adults is low (~5%), whereas in

children under one year it reaches 95%. In 2022, 1.1

million deaths related to CHB outcomes, such as liver

cirrhosis and HCC, had been recorded worldwide [6].

Serological markers related to HBV infection in-

clude viral antigens HBsAg and HBeAg and antibod-

ies – anti-HBs, anti-HBe, and anti-HBc (IgM and IgG

classes). These markers allow for the identification of

patients with HBV infection as well as the diagnosis

of CHB and the differentiation of the clinical phases

of the disease, being also important for monitoring of

the effectiveness of antiviral therapy [7].

HBsAg can be detected in the blood of the pa-

tients during both acute infection and CHB. HBsAg is

located at the surface of the virions and circulates in

the blood as a part of subviral particles of various

shapes. Quantitative assessment of HBsAg levels as a

marker of viremia is important for monitoring of the

response to antiviral therapy. Anti-HBsAg antibodies

exert virus-neutralizing activity and provide protec-

tion against HBV infection [8]. HBsAg seroconversion,

defined as the lack of detectable HBsAg (<0.05  IU/ml)

and emergence of anti-HBs antibodies (>10  IU/ml) is

a crucial endpoint of CHB functional cure, implying

(in rare cases) a long-term decline in viral replication

and immunologically controlled infection. However,

in some cases occult HBV infection has also been de-

scribed, when patients with undetectable HBsAg in the

blood serum still have viral replication and inflamma-

tion in the liver, which represents an increased risk

of developing HCC and infection reactivation [9].

HBV DNA detection in the blood is the most rel-

evant and accurate marker of viral replication, which

is also widely used in HBV infection diagnostics.

Serum DNA level in CHB patients correlates with the

risk of liver damage progression. Assessment of se-

rum DNA levels is used to determine the need for

antiviral therapy and for monitoring of treatment ef-

fectiveness [7].

cccDNA is a stable form of HBV DNA that per-

sists as a viral mini-chromosome capable of binding

histones to form a nucleosome [10]. Up to 50 copies

of cccDNA are detected in the nuclei of HBV-infected

human hepatocytes. Due to the presence of the nu-

clear pool of cccDNA and the lack of approved drugs

that can directly block or destroy cccDNA, viral repli-

cation resumes following therapy cessation [11].

Development of new approaches to the treat-

ment of CHB requires adequate in  vitro and in  vivo

models reproducing all of the described features of

HBV infection, which, however, is challenging. For

example, HBV exhibits strict species tropism partially

determined by the species specificity of the recep-

tor NTCP [12]. Furthermore, development of produc-

tive HBV infection requires several host intracellular

factors, additionally limiting the range of biological

species suitable for infection modeling [13]. Apart

from searching for the species susceptible and per-

missive to HBV, studies with other Hepadnaviridae,

such as duck hepatitis  B virus (DHBV), woodchuck

hepatitis virus (WHV), and woolly monkey hepati-

tis B virus (WMHBV), are underway. This review is

aimed to describe the current in  vivo and in  vitro

models used to study HBV and related viruses as well

KOLYAKO et al.1506

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

as for testing of CHB-specific antiviral therapies. In

addition, we paid close attention to the stages and

features of HBV infection in humans that can be re-

produced by each model, which, we believe, could

help to develop next-generation drugs aiming at a

functional cure of CHB.

In vivo HBV INFECTION MODELS

Naturally susceptible animals. Chimpanzees

(Pan troglodytes) are the only non-human primates

fully susceptible to HBV [14]. They reproduce acute

HBV infection and can develop its chronic and per-

sistent forms, resulting in less pronounced pathologi-

cal changes in the liver as compared to humans [15].

Since 1972, when hepatitis B development in chim-

panzees following inoculation of viral material ob-

tained from humans was first reported [16], this spe-

cies has been used as an animal model to investigate

both HBV pathogenesis and immune response against

HBV. For instance, cross-reactive immune response to

HBsAg derived from the diverse viral subtypes has

been proven using chimpanzees [17]. Delayed-type

hypersensitivity was demonstrated in chimpanzees

following subcutaneous HBsAg inoculation, thereby

proving HBsAg immunogenicity [18]. Hence, these

studies laid the foundation for the development of

the first HBV vaccines, efficacy and safety of which

were also tested in chimpanzees. First-generation

vaccines were made by purifying HBsAg from the se-

rum of asymptomatic HBV carriers [19]. The resulting

preparation became the first human serum-derived

vaccine licensed in the United States [20]. However,

due to the concerns about the residual presence of

HBV and other viruses in the biomaterial, alternative

vaccine types have been later developed and tested

on chimpanzees, including the pre-S2 region-encoded

synthetic peptide [21], human hepatoma cell line PLC/

PRF/5-derived HBsAg [22], DNA vaccines [23], etc. Fur-

thermore, the currently used vaccine consisting of re-

combinant HBsAg produced in yeast (Saccharomyces

cerevisiae), has been tested in the chimpanzee model

[24]. Studies using chimpanzees revealed the crucial

role of proinflammatory cytokines in the clearance of

HBV during acute infection, achieved without exten-

sive destruction of hepatocytes [25].

Due to the ethical concerns and high cost, labora-

tory use of chimpanzees is limited, which has led to

the search for new models of HBV infection primarily

among other primates. However, the species-specific

differences in the amino acid sequence of the NTCP

orthologs make Old World primates insusceptible to

HBV. To skip the viral entry stage, a plasmid encod-

ing HBV genome dimer was directly injected into the

liver of Barbary macaques (Macaca sylvanus). Two

days after the injection and several weeks onward,

the presence of HBsAg and HBV DNA was detected in

the serum and liver tissue of the animals. Subsequent-

ly, the analysis of animal serum samples revealed the

HBV-like viral particles along with pathological liver

changes resembling those observed during hepatitis B

infection in humans. Hence, the obtained data demon-

strate that the HBV replication could occur in the

Barbary macaque hepatocytes [26].

In 2013, HBV DNA as well as HBsAg and HBcAg

were detected in the blood serum and liver tissue, re-

spectively, of cynomolgus macaques (Macaca fascicu-

laris) from the island of Mauritius, whereas no patho-

logical changes in the liver tissue were observed [27].

However, it was later reported that the virus isolated

during this study was unable to induce HBV infection

in the same macaque species [28].

The northern treeshrew (Tupaia belangeri) is

an HBV-susceptible small rodent-like mammal that

is phylogenetically more closely related to primates

[29]. Adult animals directly infected with HBV ex-

hibited a self-limiting acute infection with increased

HBsAg levels in the serum 2-3 weeks post-infection

along with the rapidly produced anti-HBeAg anti-

bodies [30]. Injection of the virus into neonatal ani-

mals caused chronic infection lasting over 48-weeks.

cccDNA has been detected in the liver of northern

treeshrews with CHB along with pathological changes

in the liver morphology, which developed at a slower

rate than in humans [31]. Nonetheless, chronic HBV

infection in treeshrews could result in HCC develop-

ment [32], which is not typical for some other animal

models of HBV infection. Additionally, immunohisto-

chemical staining revealed the presence of HBcAg in

hepatocytes during both acute and chronic infection,

confirming viral replication process [31]. Studying of

HBV using treeshrews is hampered by the difficulty in

selecting adequate species-specific reagents and pro-

tocols, low viral loads, and the need to infect neona-

tal animals, while the incidence of CHB development

is low (13%) [30].

Genetically modified animals. Susceptibility to

HBV depends on the species-specific host factors, pri-

marily the sequence of NTCP orthologs. Human NTCP

gene (hNTCP) transduction into mouse, rat, and dog

hepatocytes followed by HBV infection ensures virus

entry into the cells but does not result in effective

viral replication, suggesting that species-specific in-

tracellular factors affect the HBV replication cycle.

Hepatocytes derived from cynomolgus macaques, rhe-

sus macaques, and pigs, after transduction of hNTCP,

supported complete HBV replication cycle compara-

ble to that in human hepatocytes, thereby opening

the way to use immunocompetent macaques in stud-

ies of HBV infection, immune response, and viral

pathogenesis [33].

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Due to the general similarity between the hu-

man and macaque immune systems, rhesus macaques

(Macaca mulatta) have several advantages over oth-

er HBV infection models [34]. Inoculation of rhesus

macaques with the hNTCP-encoding adenoviral vec-

tor followed by infection with HBV [28] led to mod-

erate viremia. However, no chronic infection devel-

oped in this model. Immunohistochemical staining

of the liver sections revealed the presence of HBcAg

solely inside the nuclei of 0.5-1.0% hepatocytes [28].

In order to achieve chronic infection, immunosup-

pression was additionally induced in infected rhesus

macaques, which resulted in persistent 20-week long

HBV replication identified by the presence of HBsAg

and HBeAg in the blood as well as HBV DNA in liver

biopsies. However, after cessation of immunosuppres-

sion, immune-mediated virus elimination occurred in

most animals with emergence of serum anti-HBs and

anti-HBc IgG antibodies, as well as CD8

and CD4

T cell activation [35]. Thus, the genetically modified

hNTCP-expressing rhesus macaques could maintain

prolonged HBV infection subsequently resulting in

specific immune-related virus elimination.

Surrogate models. Duck hepatitis B virus. The

studies with other members of Hepadnaviridae fam-

ily were conducted in parallel with the search for

HBV-susceptible animal species. In 1980, a virus later

named duck hepatitis  B virus (DHBV) was discovered

in the blood serum obtained from domestic ducks

(Anas domesticus). Viral DNA was detected mainly

in the liver, suggesting hepatospecificity of the virus.

Electron microscopy analysis of the purified virus re-

vealed particles (40  nm in diameter) similar to those

of other members of the Hepadnaviridae family [36].

Similar to HBV, the DHBV genome is represented by

rcDNA [37]. The DHBV-infected ducks were used as

a model for testing of antiviral drugs such as poly-

merase inhibitors [38] and nucleocapsid assembly

inhibitors [39]. Although ducks offer a relatively con-

venient model for laboratory use, extrapolation of the

research data to humans is complicated because of the

differences in the genome sequence and pathogenesis

between DHBV and HBV. In particular, nucleotide se-

quence of the DHBV genome is only 40% homologous

to that of HBV [40]. Moreover, specific virus receptors

also are different: the entry receptor for DHBV is car-

boxypeptidase D [41], which does not allow to study

the drugs targeting the interaction between HBV and

its receptor. In addition, no correlation was found be-

tween chronic hepatitis and the development of HCC

in various duck species infected with DHBV [42].

Woolly monkey hepatitis  B virus. Besides HBV,

Orthohepdnavirus genus also includes woolly monkey

hepatitis  B virus (WMHBV), identified in the blood of

common woolly monkeys (Lagothrix lagotricha). The

WMHBV amino acid sequence is 44% homologous to

that of HBV, however the HBcAg gene which is pre-

sumably conserved among the Hepadnaviridae family,

has 85-86% and 75-77% homology to the HBV HBcAg

in amino acid and nucleotide sequence, respectively.

The WMHBV causes either acute (fulminant) or chron-

ic infection in common woolly monkeys [43]. Other

primates were also experimentally infected with

WMHBV. HBsAg and WMHBV DNA were detected in

black-handed spider monkey (Ateles geoffroyi) blood

serum for 4-6 weeks after infection, while no patho-

logical changes were detected in liver biopsies [43].

In order to establish chronic WMHBV infection, neona-

tal black-handed spider monkeys were infected with a

WMHBV clone, which did not lead to the development

of a chronic disease [44]. As common woolly monkeys

and black-handed spider monkeys are endangered

species, ethical reasons hamper their large-scale re-

search use, which motivated researchers to use a re-

lated primate species, squirrel monkeys (Saimiri sciu-

reus), as an alternative. Infection of squirrel monkeys

with WMHBV-containing biomaterial lead to acute in-

fection with viremia that lasted for 4 weeks. To pro-

long the infection, the animals were inoculated with

the adeno-associated virus (AAV8) encoding infectious

WMHBV genome, which resulted in viremia lasting

32 weeks. In all neonatal squirrel monkeys infected

with WMHBV viremia lasted for 3 months, followed

by virus elimination [45]. Thus, despite the absence

of chronic infection, squirrel monkeys infected with

WMHBV can be used as a surrogate model of HBV

infection.

Woodchuck hepatitis virus (WHV). WHV also be-

longs to the genus Orthohepdnavirus. Along with

general similarity in the genome and virion struc-

ture (WHV genome has 62-70% nucleotide sequence

homology to HBV), WHV and HBV exhibit antigenic

cross- reactivity. Also, these viruses have the same

timeline of viral antigens appearance in the liver and

blood after infection, which indicates the similarity

of the stages of infection course [46]. In adult wood-

chucks, acute WHV infection may result either in

complete virus elimination or progression into chron-

ic form. Inneonatal woodchucks, the infection mainly

becomes chronic [47]. Chronic WHV-infection leads to

exhaustion of the WHV-specific T-cell response as well

as to low interferon production by hepatocytes [48].

WHV was first detected in the tissues of dead

woodchucks (Marmota monax), which showed signs

of HCC development [47]. Similar to HBV, WHV is an

oncogenic virus [49], with HCC developing in 90% of

cases of verified chronic WHV infection [49]. There-

fore, high HCC incidence allows to use woodchucks

as a convenient model in studies of viral oncogenesis.

The ability of the WHV genome to integrate into the

cell genome and cause increased proliferation of he-

patocytes was studied using the marmot model [50].

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Woodchuck models have been useful for preclinical

testing of antiviral drugs, including nucleoside ana-

logues [51]. In 2011, Himalayan marmots (Marmota

himalayana) were also shown to be susceptible to

WHV, expanding the panel of available surrogate

models of HBV infection [52]. However, susceptibility

to WHV varied between different animals [53]. Appli-

cability of woodchucks as an HBV infection model is

limited due to the difficulties in laboratory housing

and care of these animals and limited availability of

reagents for the immune response studies.

Mouse models. For biomedical research, mice are

the most common and easy-to-use model, with stan-

dardized methods and reagents available for a wide

range of experiments. Because mice are normally nei-

ther susceptible nor permissive to HBV, gene modifi-

cation and human tissue transplantation are used to

generate mouse models of HBV infection.

Transgenic mice. Due to the development of

the embryonic microinjection technology, transgen-

ic mouse models have been created, which carry in

their genome either individual HBV genome regions

encoding HBsAg [54], HBeAg [55], and HBxAg [56] or

full-length HBV genome [57]. The hepatocyte-specif-

ic promoters such as the mouse metallothionein or

albumin promoters were used to control HBV gene

expression in the mouse liver [58].

HBsAg-producing transgenic mice were used for

the modeling of the asymptomatic HBV carriage [54]

as well as for the validation of the indirect oncogenic

effect of HBsAg [59]. Moreover, the oncogenic effect

of HBxAg was demonstrated in transgenic mice, as

production of HBxAg was directly associated with his-

topathological changes in the liver [56]. HBV vertical

transmission was shown in transgenic female mice

bearing complete HBV genome. In addition, it has

been shown that HBeAg could induce the production

of the regulatory peptide PD-L1 by liver macrophages.

Production of PD-L1 leads to the decrease in CD8

cell proliferation [60], resulting in HBV persistence

in the transgenic mouse offspring [61]. A disadvan-

tage of transgenic mouse models is that the proteins

produced from the transferred genes are not recog-

nized as foreign by the immune system of the ani-

mal, which hinders the assessment of the immune re-

sponse. Furthermore, there is no recirculation of the

assembled viral particles into hepatocytes of transgen-

ic mice bearing complete HBV DNA sequence, as the

mouse NTCP ortholog is unable to facilitate HBV entry

into cells. The transgenic mouse model is not relevant

for testing of therapeutic drugs aimed at blocking or

destroying cccDNA, because this form of HBV genome

is not formed in mice. The virions assemble due to

the expression of linear genomic sequences of the vi-

rus integrated into the mouse genome, which makes

their complete elimination impossible [62].

Mouse models with invivo transfection. To avoid

genetic aberrations caused by the HBV genome inte-

gration into the mouse genome, models of episomal

HBV assembly have been developed. Currently, two

methods are used to deliver HBV genome into the

mouse hepatocytes either as HBV-encoding plasmids

or as recombinant cccDNA (rcccDNA): high-pres-

sure hydrodynamic injection-based HBV transfection

(HDI-HBV) and transduction using adeno-associated

virus (AAV) or adenovirus (Adv) vectors, bearing HBV

genome. The HDI-HBV results in transfection of 10%

hepatocytes, whereas adenoviruses provide transduc-

tion of 90% hepatocytes. The drawbacks of such ap-

proaches are as follows: (i) lack of the infection stage

and full viral replication cycle, as the “surrogate” HBV

cccDNA is formed in the transduced mouse cells, from

which viral transcripts are produced; (ii) non-physi-

ological viremia levels – tens of times higher than

in natural infection; (iii)potential for development of

the robust immune response both against viral com-

ponents due to the high level of virion production

and against adenovirus components; (iv) tissue non-

specificity of HDI-HBV, as along with hepatocytes the

viral genome may potentially enter other cell types

[63]. Since hepatotoxic effects are observed upon us-

ing the plasmids/rcccDNA at high doses in HDI-HBV or

when inoculating >10

adenovirus particles encoding

the HBV genome (AdHBV), 10μg DNA in HDI-HBV and

up to 10

AdHBV should be used in in  vivo experi-

ments [64, 65].

Genetic background of the mice used in the ex-

periments also affects the course of HBV infection.

In particular, BALB/c mice after HDI-HBV (10  μg of

plasmid in a volume equivalent to 8% of mouse

body weight) are predisposed to HBV elimination

with production of anti-HBsAg antibodies within 14

days post-injection, whereas in C57BL/6 mice HBsAg

was detected for 35 days after plasmid injection [66].

The main drawback of the described mouse models

is the absence of cccDNA. To overcome this obstacle,

a precursor recombinant cccDNA plasmid (prcccDNA)

was synthesized and transduced using Adv into the

C57BL/6 and Alb-Cre Tg mice (C57BL/6-Tg [Alb-cre]

21Mgn/J). Recombination of prcccDNA occurred in

the nuclei of the transduced hepatocytes resulting in

the production of rcccDNA. Viremia in C57BL/6 mice

was not observed following HDI-HBV, whereas HBcAg

was detected at low levels and located primarily in

the hepatocyte nuclei. At the same time, in Alb-Cre

Tg mice rcccDNA was detectable for 62  weeks accom-

panied by the pronounced viremia as evidenced by

the detection of HBcAg and HBsAg in the blood. In-

creased PD-L1 production in the liver also resulted in

the decrease in specific T-cell response against the rc-

ccDNA-positive hepatocytes, which contributed to the

rcccDNA persistence [67].

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Fig. 1. Stages of HBV infection and related pathogenesis reproduced in the in vivo models. The animal species and relevant

viruses as well as recombinant vectors used for each model are presented. Designations: DHBV, Duck Hepatitis B Virus;

WHV, woodchuck hepatitis virus; WMHBV, woolly monkey hepatitis B virus; AAV, adeno-associated virus. Species-specific

descriptions: P.t., Pan troglodytes; T.b., Tupaia belangeri; M.f., Macaca fascicularis; M.m-a, Macaca mulatta; M.m., Mus mus-

culus; A.d., Anas domesticus; M.m-x, Marmota monax; M.h., Marmota himalayana; L.l., Lagothrix lagotricha; A.g., Ateles

geoffroyi; S.s., Saimiri sciureus). cccDNA, covalently closed circular DNA; HCC, hepatocellular carcinoma.

To assess the efficacy of novel HBV treatments

in vivo, an episomal vector based on the adeno-as-

sociated virus AAV9-HBsAg was developed. AAV9-HB-

sAg serves as a surrogate for cccDNA and contains a

partial HBV sequence under the control of the hep-

atospecific promoter. Inoculation of AAV9-HBsAg into

NOD-SCID IL2rg

−/−

mice resulted in HBsAg secretion

lasting for 7 weeks. Subsequently, the AAV8-HBsAg

was also used in the cynomolgus macaque (Macaca

fascicularis) model to test the HBV genome-specific

engineered nuclease (ARCUS-POL) [68].

Thus, a wide range of in vivo HBV infection

models designed in diverse animal species is current-

ly available, each of which (except for chimpanzee

model) only partially reproduces the course of human

HBV infection. The advantages and drawbacks of such

models are shown in Fig. 1.

Xenograft-based mouse models. Transgenic and

transduced mice models do not fully reproduce the

natural course of HBV infection in vivo; moreover,

many antiviral drugs cannot be tested using such

models. To reproduce the complete HBV replication

cycle in mice, the Trimera model was developed. For

this, the immunocompetent CB6F1 mice were exposed

to lethal gamma irradiation followed by inoculation

with bone marrow cells derived from SCID/NOD mice.

Fragments of human HBV-infected liver tissue were

then transplanted under the subrenal capsule or into

the auricle of the animals. HBV DNA was detected in

the serum of the CB6F1 mice bearing human liver

KOLYAKO et al.1510

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

xenografts from day 8 to day 25 post-transplantation

and declined afterwards. A similar infection profile

was achieved using the Trimera model in the immu-

nodeficient BNX mice. cccDNA was detected in the liv-

er grafts indicating viral replication inside the human

hepatocytes. The Trimera model was used to evaluate

the efficacy of antiviral drugs including polyclonal

anti- HBsAg antibodies, reverse transcriptase inhib-

itors, and nucleoside analogues [69]. Another mod-

el was also created using nude (nu/nu) mice, which

were subcutaneously injected with the HepAD38 cells

bearing complete HBV genome under the control of

tetracycline repressor. Viral replication was regulated

by the presence of tetracycline (tet) in drinking wa-

ter. In the absence of tet, the mice developed viremia

lasting 35 days after infection, and cccDNA was de-

tected in the liver. Viral load decreased after adding

tet to drinking water and relapsed after tet removal

[70]. The developed model was used to test combi-

nation therapy against HBV in vivo [71]. Long-term

HBV viremia was achieved using a transplantation

model based on mice with combined RAG-2 deficiency

(RAG-2M). For this, a primary human hepatocyte cul-

ture was immortalized by transduction with the SV40

T-antigen followed by transfection with the full-length

HBV genome and obtaining monoclonal cell line

(IHBV6.7). The latter was transplanted into the mice

via intrasplenic injection. Although the transplanted

IHB6.7 cells comprised as few as 1% of the mouse

hepatocyte mass, viremia lasted for at least 5 months

post-transplantation [72]. The ability to not only in-

tegrate into, but also partially repopulate the mouse

liver with human hepatocytes (up to 15% mouse liv-

er) was first demonstrated in the uPA/RAG-2 model,

using immunodeficient RAG-2 knockout mice crossed

with the uPA transgenic mice. Overexpression of the

urokinase-type plasminogen activator (uPA) gene in

mouse hepatocytes resulted in neonatal hemorrhage

and liver necrosis. Following human hepatocyte xeno-

transplantation, mouse hepatocytes were replaced in

the liver by human hepatocytes. Infection of the uPA/

RAG-2 mice with serum samples derived from HBV

carriers resulted in viremia and detection of HBsAg

for 8 weeks following infection [73]. Another mod-

el was developed based on SCID mice homozygous

for the Alb-uPA transgene [74]. Infection of the xeno-

transplanted SCID Alb-uPA mice with serum samples

derived from HBV carriers resulted in viremia that

was detected at week 2 after infection and persisted

for 12 weeks. It was also shown that HBV particles

derived from HepG2 cells bearing the full-length vi-

ral genome were able to infect SCID Alb-uPA mice.

However, the viral load detected from week 4 was

significantly lower compared to infection with blood

serum samples. The drawback of the Alb-uPA SCID

model was the lack of the control over uPA expres-

sion, the need for transplantation into neonatal mice,

and increased risk of hemorrhage [75]. Therefore, an

alternative Fah

−/−

Rag-2

−/−

Il2rg

−/−

mouse model was

created using immunodeficient animals with deleted

gene encoding fumarylacetoacetate hydrolase (Fah).

Loss of the latter results in accumulation of toxic

tyrosine metabolites in mouse hepatocytes causing

their death, which promotes liver repopulation with

the xenograft hepatocytes. Furthermore, the toxicity

level could be controlled by the orally administered

2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexane-

dione (NTBC). Following human hepatocyte trans-

plantation, the mouse liver comprised as many as

20% of the xenograft hepatocytes, and repopulation

success rate ranged from 45 to 95% [76]. Mice with

high proportion of liver repopulation were infected

with HBV. From the 2nd to the 7th week of follow-up,

cccDNA was detected in liver samples and viremia

was confirmed with the peak of the viral load detect-

ed at the 6th week. Immunocytochemical staining for

HBcAg demonstrated that the infection proceeded ex-

clusively in the transplanted human hepatocytes, with

HBcAg accumulation observed in both the nuclei and

cytoplasm [77]. The model was used to evaluate the

efficacy of novel antiviral drugs [78].

Assessment of the immune response in xenograft

mouse models is complicated, as it requires the use

of immunocompromised animals. To overcome this

limitation, additionally to human hepatocytes, mice

were transplanted with human immune system (HIS)

elements. In particular, the neonatal A2/NSG mice ex-

pressing human HLA-A2 gene were inoculated with

donor-derived CD34

hematopoietic stem cells (HSC)

and human hepatocyte progenitor cells (Hep). This

resulted in the development of a functional human

immune system along with liver repopulation. In this

process, HLA-A2 expression increased the selection of

T cells producing only human MHC. To promote liv-

er repopulation with human cells, mouse hepatocyte

death was selectively induced by anti-murine Fas an-

tibodies. It was found that the A2/NSG-hu HSC/Hep

mice developed low viremia over 12 weeks after HBV

infection. The immune response was characterized

by the increased production of IFN-γ, IP10, and IL-6

along with the low levels of IL-10 and IFN-α, as well

as the presence of anti-HBsAg antibodies and HBV-spe-

cific CD8

T cells. While examining the liver tissue

in HBV-infected mice, increased collagen deposition

was described, which is typical for HBV-induced tis-

sue fibrosis during CHB [79]. For dual humanization,

the BALB/c Rag-2

−/−

Il2rg

−/−

Sirpa

NOD

 Alb-uPA

tg/tg

mice

were also used for transplantation of human hema-

topoietic stem cells. The resulting model (HIS-HUHEP)

demonstrated high human hepatocyte engraftment

rate (about 20-50% of mouse liver was replaced with

human cells) [80]. Infection of HIS-HUHEP mice with

HEPATITIS B VIRUS INFECTION MODELS 1511

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

HBV showed that the viral load and accumulation

of T cells, Kupffer cells and natural killer cells (NK

cells) in the liver correlated with the administered

HBV dose. However, the disease progressed to chron-

ic course without the development of liver fibrosis.

Itwas also shown that the liver PD-L1 protein expres-

sion in infected animals was elevated in comparison

with intact mice. It has been shown previously that

PD-L1 expression leads to T-cell exhaustion, which is

one of the causes of CHB [81]. Another alternative

model is based on Fah

−/−

Rag-2

−/−

IL-2Rγc

−/−

SCID (FRG)

mice transplanted with human bone marrow-derived

stem cells (hBMSCs). After hBMSC-FRG animals were

infected with HBV, viral DNA and antigens were de-

tected in serum at relatively high levels for 56 weeks.

Immunohistochemical staining of the liver sections

for human albumin, HBsAg, and HBcAg revealed

that only human hepatocytes were infected with HBV.

The percentage of infected hepatocytes increased up

to 80% over time. Analysis of mouse liver sections

also showed increased levels of proinflammatory

cytokines, human chemokines, as well as of hCD3,

hCD86, and NK cells, suggesting an active immune

response. Moreover, the humoral response was also

characterized by the presence of specific IgG against

HBsAg and HBcAg, which, however, declined sharply

at the week 32. Pathological liver changes typical to

chronically infected patients were observed through-

out the experiment, which included lobular inflamma-

tion, lymphoid aggregates, and duct damage, as well

as lymphocyte infiltration from the portal vein. At the

later stages, signs specific to the developing cirrhosis

were observed: collagen accumulation, fibrous tissue

hyperplasia, and abnormal liver lobe structure [82].

Xenograft mouse models played an important role

in investigating HBV pathogenesis in vivo as well as

efficacy assessment of antiviral therapy. The double-

humanized mice also allowed to assess the impact

of chronic HBV infection on the development of im-

mune response. However, use of such animal models

is complicated by the labor-intensive process of their

obtaining, high cost, and risk of sudden death. The

advantages and drawbacks of the described models

are summarized in Table S1 (Online Resource 1).

In vitro HBV INFECTION MODELS

Primary human hepatocytes. Primary human

hepatocytes are derived from the livers of organ do-

nors, tissues after liver surgery including resections,

cadaveric material, and liver biopsies [83]. Due to

the natural susceptibility to HBV infection, human

hepatocyte culture serves a reference model for HBV

research. Hepatocytes fully support HBV replication

cycle and, moreover, possess a functional intracellular

innate immunity allowing for studies of the innate

antiviral immune response against HBV [84]. How-

ever, primary hepatocytes undergo rapid phenotypic

changes during in  vitro culturing [85]. Initially, pri-

mary hepatocytes could be maintained in  vitro for up

to one month. Modifications in culturing conditions

allowed to extend hepatocyte viability up to 2 months

without affecting their metabolism [86], which is still

insufficient for CHB modeling. Culture media supple-

mentation with dimethyl sulfoxide (DMSO) has been

shown to promote hepatocyte differentiation, leading

to a significant increase in HBV replication level [87].

HBV viral particle and protein production levels vary

between the primary hepatocyte cultures obtained

from different donors [88]. Thus, although primary

human hepatocytes support full HBV replication cycle,

use of this model is limited due to the low acces-

sibility and heterogeneity of the biomaterial, which

profoundly complicates their large-scale application.

Human hepatoma cultures. HepG2 and Huh-7

are the most widely used human hepatoma cell lines

due to their in  vitro stability and ease of cultivation.

However, due to the lack of surface hNTCP, they are

not susceptible to HBV infection. The hepatoma cells

can only be used as HBV infection models after their

modification by HBV genome transfer or by hNTCP

expression followed by HBV infection.

Huh-7 cells, isolated from hepatocellular carcino-

ma in 1982 [89], were shown to be a useful in  vitro

model for studying of HBV replication cycle. After

Huh-7 cells were transfected with a plasmid encod-

ing HBV DNA genome dimer, HBsAg and HBeAg were

detected in the culture medium for 25 days. More-

over, the presence of viral particles was confirmed

in culture medium at day 20 after transfection [90].

The Huh-7 cell line was used as a model to investi-

gate the molecular mechanisms underlying HBV rep-

lication [91, 92] including virion assembly and mat-

uration [93], impact of the immune factors on viral

replication [94] and efficacy assessment of antiviral

agents [95, 96] including novel therapeutic approach-

es based on TALENs- and CRISPR/Cas9 cccDNA cleav-

age systems [97].

HepG2 cells were isolated from the hepatocellular

carcinoma of a 15-year-old patient [98]. After transfec-

tion of HepG2 cells with a plasmid encoding an HBV

DNA genome dimer, the HepG2.2.15 monoclonal cell

line with genome-integrated HBV DNA was obtained

[99]. Infectivity of the viral particles isolated from

HepG2.2.15 cells was demonstrated in the chimpanzee

model [100]. Additionally, HepG2.2.15 cells allowed to

identify the potential molecular targets for CHB ther-

apy [101]. In 1997, the HepAD38 cell line was derived

from HepG2 cells after transfection with the plasmid

encoding HBV genome under the control of tetracy-

cline-responsive promoter [102]. Later, the modified

KOLYAKO et al.1512

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

cell lines, HepDE19 and HepDES19, were obtained,

in which viral particle production was tetracycline-

depended [103]. These cell lines were used to screen

potential inhibitors of cccDNA formation [104].

HepaRG cells were derived from the hepato-

cellular carcinoma cells of a hepatitis C-infected

patient. After differentiation by adding DMSO and

corticosteroids to the culture medium HepaRG cells

morphologically and functionally resembled primary

hepatocytes and were successfully infected with HBV.

HBsAg, HBcAg, and HBV DNA were detected in the

culture medium after infection [105]. The presence of

cccDNA in the infected cells was later confirmed in

a 50-day experiment. However, susceptibility of the

HepaRG cells is strictly dependent on cell differen-

tiation, which requires extended period of time to

achieve (2 weeks for proliferation and 2 weeks for

differentiation in the culture medium supplemented

with DMSO). Moreover, the HepaRG cell line consists

of differentiated and polarized hepatocyte-like cells

and biliary-like epithelial cells, which results devel-

opment of the infection in islands of differentiated

cells [106]. Additionally, prolonged exposure to DMSO

induces cell death, which also affects the HBV infec-

tion characteristics [107].

Upon discovering hNTCP as a functional receptor

of HBV [12], the research focus was directed towards

the generation of HBV-susceptible cell lines by hNTCP

overexpression in cells. It was experimentally con-

firmed that hNTCP-expressing HepG2 and Huh-7 cell

lines (HepG2-NTCP and Huh7-NTCP, respectively) are

susceptible to HBV purified from serum samples of in-

fected patients, as well as the virus isolated from cul-

ture medium. The HepG2-NTCP and Huh7-NTCP cells

support complete HBV replication cycle, from viral

entry to cccDNA formation. Due to the ease of their

production, these cell lines have been widely used for

testing and development of antiviral drugs [108, 109].

However, HBV infection efficiency in HepG2-NTCP and

Huh7-NTCP cell lines varies. Although hNTCP expres-

sion allows HBV to enter Huh7-NTCP cells, further viral

replication occurs in a smaller proportion of cells in

comparison with the HepG2-NTCP cell line [110]. The

current HepG2-hNTCP models allow for infection of

up to 50-60% of cells (https://www.abmgood.com/ntcp-

stable-hepg2-cell-line.html, accessed 18.04.25). Despite

this, virus particle production by the HepG2-NTCP cell

line is low. In order to create a cell culture with high

virus yield, the monoclonal HepG2-NTCP-sec

culture

with low proliferative activity was selected, in which

more than 90% of the cells remained infected for

4weeks after HBV infection without a decrease in the

levels of detectable HBV DNA [111].

Overall, human hepatoma cells represent a useful

and convenient tool for researchers, but their wide-

spread use is profoundly limited by the fact that

HBV infection is transient, without significant spread

among new cells. Moreover, the hepatoma cells differ

from the primary hepatocytes in multiple physiolog-

ical functions and mechanisms, that complicates the

data interpretation. Oncogenic origin of such cell lines

complicates their use in investigating the mechanisms

behind the HBV-induced carcinogenesis.

Animal cell lines. Being susceptible to HBV, the

northern treeshrew played a crucial role not only in

the in  vivo but also in in  vitro studies of HBV infec-

tion. The infection of primary treeshrew hepatocytes

resulted in HBV DNA and RNA production lasting

for 12 days as well as in the detection of HBsAg and

HBeAg in the culture medium for up to 5-6 days [30].

Later, accumulation of cccDNA was also demonstrat-

ed. Furthermore, treeshrew hepatocytes were shown

to be susceptible to WMHBV, with higher efficien-

cy of viral replication compared to HBV [112]. The

HBV-infected treeshrew hepatocytes were used as an

in  vitro model for efficacy assessment of antiviral

agents [113] and in studies of virus-cell interactions

[114]. This model was used to prove that the HBV

pre-S1 domain enables HBV entry, which occurs in a

species-specific manner [115]. Treeshrew hepatocytes

were used to assess the infectivity of viral particles

produced by human hepatoma cell lines [116]. Tree-

shrew primary hepatocytes are also a more readily

available alternative to human primary hepatocytes.

This allowed their use in the development of a xe-

nograft mouse model, which was subsequently suc-

cessfully infected with HBV [117] and used to test a

potential inhibitor of HBV entry [118]. In addition, the

treeshrew xenograft mouse model helped to describe

the emerging hepatocyte proliferation, which resulted

in the decreased population of the cccDNA-containing

cells [119].

Among other studied animals, the common mar-

moset (Callithrix jacchus) is a promising animal mod-

el of HBV infection. Although common marmoset he-

patocytes are not directly susceptible to HBV infection,

it has been shown that the Adv-mediated delivery of

the full-length HBV genome to a primary hepatocyte

culture resulted in viral replication with detection of

cccDNA and its derivatives, which was followed by

HBV particle formation, confirming permissiveness of

the common marmoset cells to HBV [120]. Recently it

has been found that common marmoset hepatocyte

resistance to HBV is associated with amino acid differ-

ences between common marmoset NTCP and hNTCP

at two positions directly involved in HBV binding.

In order to overcome this obstacle, a chimeric HBV/

WMHBV pre-S1 virus was constructed and isolated.

This virus successfully entered and replicated in

common marmoset hepatocytes, as evidenced by the

detection of HBeAg in the culture medium and the

presence of HBV cccDNA in cells [121]. Taken together,

HEPATITIS B VIRUS INFECTION MODELS 1513

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

these results suggest high potential for further use

of common marmoset hepatocytes in HBV research.

However, currently common marmoset hepatocyte

cell lines are not yet available.

Primary hepatocytes from woodchucks and ducks

have also been used for in  vitro studies. When prima-

ry woodchuck hepatocytes were infected with WHV,

cccDNA formation was detected on day 2 post-infec-

tion followed by the detection of DNA fragments for

7-10 days, indicating active WHV replication  [122].

HBV-infected woodchuck hepatocytes served as a mod-

el for efficacy assessment of antiviral drugs, such as

DNA synthesis inhibitors [123]. Due to the relative ease

of culturing, primary duck hepatocytes were more

widely used [124]. The DHBV-infected duck hepato-

cyte culture allowed to explore reverse transcription

activation [125] and cccDNA formation from rcDNA

[126], as well as nucleocapsid assembly [127]. How-

ever, translational value of the obtained data is ham-

pered by the above-mentioned genomic differences

between HBV and DHBV.

Hence, hepatocyte cell cultures of various origins

are widely used in the studies of various stages of

HBV replication cycle, as well as in testing of potential

antiviral drugs. However, similar to the in  vivo mod-

els, each type of cell cultures has certain limitations

that must be taken into consideration while making

the decisions regarding their use in experiments. The

hepatocyte culture-based models described here are

summarized in Table S2 (Online Resource 1).

CONCLUSION

Undoubtedly, HBV infection poses a serious global

health threat. Relevant and accessible models of viral

infection are required to investigate the mechanisms

underlying HBV pathogenesis, identify new antiviral

targets, as well as to test novel antiviral vaccines and

therapeutics. Here, we review most of the currently

available models used to study HBV infection.

Among the in vivo models, the chimpanzee has

historically served as a reference research model due

to the natural susceptibility to HBV and phylogenetic

proximity to humans. However, high cost and difficul-

ty in maintenance in laboratory conditions limit the

widespread use of chimpanzees. Among the in  vitro

models, primary hepatocyte cultures serve as a stan-

dard reference model. Both models are virtually inap-

plicable for large-scale studies due to ethical concerns

and limited availability.

Ideally, a universal HBV model should meet the

following parameters: (i)  viral replication level similar

to the one observed in natural infection, with max-

imum infection efficiency; (ii)  recapitulate all stages

of infection development, replication cycle and im-

mune response; (iii)  be widely accessible with regard

to cost-effectiveness and availability of reagents;

(iv)  ensure data reproducibility. Owing to decades of

research, a wide range of diverse HBV infection mod-

els have been developed, but none of them fully meet

these criteria.

In this regard, modifications of the existing

in  vitro models such as HepG2-NTCP-sec

cell line

and the search for new susceptible animals could

provide most promising approaches to development

of a universal model. It is worth noting that, despite

the mentioned drawbacks, genetically modified and

humanized mice are most widely used as in  vivo

models, in part due to accessibility and availability

of the necessary reagents, particularly specific anti-

bodies. In this regard, the model using common mar-

moset hepatocytes and chimeric HBV/WMHBV pre-S1

virus undoubtedly has an advantage due to the wide

panel of available methods and reagents.

In conclusion, no universal model of HBV infec-

tion able to recapitulate all the stages of viral repli-

cation cycle and host immune responses is currently

available. Its development might be a crucial step

toward creating therapies for CHB and HBV-induced

pathologies.

Abbreviations

AAV adeno-associated virus

Adv adenovirus vector

cccDNA covalently closed circular DNA

CHB chronic hepatitis B

DHBV duck hepatitis B virus

HBV hepatitis B virus

HBeAg hepatitis B virus E antigen

HBcAg hepatitis B virus core antigen

HBsAg

hepatitis B virus surface antigen iso-

forms

HCC hepatocellular carcinoma

HDI-HBV

high-pressure hydrodynamic injection-

based HBV transfection

NTCP

-taurocholate cotransporting poly-

peptide

rcDNA relaxed circular DNA

rcccDNA recombinant cccDNA

tet tetracycline

uPA urokinase-type plasminogen activator

WHV woodchuck hepatitis virus

WMHBV woolly monkey hepatitis B virus

Supplementary information

The online version contains supplementary material

available at https://doi.org/10.1134/S0006297925602230.

Contributions

Y. V. Kolyako – wrote the initial version of the

manuscript, edited the text; A. S. Zhitkevich and

KOLYAKO et al.1514

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

D. V. Avdoshina – edited the text, created the illustra-

tions; D. Y. Tanygina and V. D. Apolokhov – created

the illustrations; T. V. Gorodnicheva – edited the text;

E. O. Bayurova – edited the text, conceptualized the

study; D. S. Kostyushev and I. V. Gordeychuk – edited

the text, conceptualized the study, funding acquisition.

Funding

The study was financially supported by the Russian

Science Foundation (grant no.25-65-00010).

Ethics approval and consent to participate

This work does not contain any studies involving hu-

man and animal subjects.

Conflict of interest

The authors of this work declare that they have

noconflicts of interest.

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