Accepted Manuscript

The Virological Aspects of Hepatitis B

Zina S. Valaydon, Stephen A. Locarnini

PII: S1521-6918(17)30036-7
DOI: 10.1016/j.bpg.2017.04.013
Reference: YBEGA 1508

To appear in: Best Practice & Research Clinical Gastroenterology

Received Date: 31 March 2017

Accepted Date: 28 April 2017

Please cite this article as: Valaydon ZS, Locarnini SA, The Virological Aspects of Hepatitis B, Best
Practice & Research Clinical Gastroenterology (2017), doi: 10.1016/j.bpg.2017.04.013.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.
 ACCEPTED MANUSCRIPT
The Virological Aspects of Hepatitis B

Zina S Valaydon1, 2,3*, Stephen A Locarnini1

1
Division of Research and Molecular Development, Victorian Infectious Diseases

PT
Reference Laboratory, Peter Doherty Institute, Parkville, Victoria, Australia.
2

RI
Department of Gastroenterology, St. Vincent's Hospital, Fitzroy, Victoria, Australia
3
Department of Medicine, Eastern Hill Academic Centre, The University of

SC
Melbourne, Parkville, Victoria, Australia.

U
AN
*Correspondence: zina.valaydon@svha.org.au
M
D
TE
C EP
AC

1
 ACCEPTED MANUSCRIPT
Abstract

Human hepatitis B virus (HBV) is a hepatotropic virus that is responsible for a

significant burden of disease, causing liver disease and hepatocellular carcinoma. It is

a small DNA virus with a replication strategy that is similar to that of a retrovirus.

HBV is prone to mutagenesis and under the influence of diverse selection pressures,

PT
has evolved into a pool of quasispecies, genotypes and mutants, which confers a

RI
significant survival advantage. The genome is small, circular, and compact but has a

complex replication strategy. The viral life cycle involves the formation of a

SC
covalently closed circular DNA (cccDNA), which is organized into a

minichromosome that is the template for the synthesis of viral mRNA. HBV DNA

U
(double-stranded linear form) can also integrate into the host genome, ensuring
AN
lifelong persistence of the virus. To date, despite great advances in therapeutics, once
M

HBV is chronically established, it is incurable. This is by virtue of many aspects of its

virological structure and viral life cycle. In this review, we aim to discuss important
D

aspects of the virology of HBV with a focus on clinical implications.

TE

Keywords: Virology Viral structure viral genome viral replication genotypes

C EP
AC

2
 ACCEPTED MANUSCRIPT
Introduction

The hepatitis B virus (HBV) is a member of the Hepadnaviridae family, a family of

small, enveloped viruses that primarily cause hepatotropic infections. Hepadnaviridae

have two species-specific genera, orthohepadnaviruses that infect mammals and

avihepadnaviruses that infect birds. Human HBV is the prototype of the

PT
orthohepadnavirus genus (1).

RI
Like all hepadnaviruses, HBV causes a hepatotropic infection and it has not been

isolated in tissues other than the liver. HBV can cause both acute and chronic

SC
infections but the virus itself is not directly cytopathic: hepatitis and any resulting

liver damage are usually mediated by the immunological response of the host to the

virus.
U
AN
HBV appears to require specific host factors for various aspects of its life cycle
M

therefore maintaining species specificity. Primarily, HBV infects humans although

infection is permissive in great apes, chimpanzees, macaques and in the laboratory

D

setting, the tree shrew (2-4). However, in experimental models, surrogate HBV-like
TE

viruses have been successfully used to address particular questions concerning

pathogenesis and replication, including ducks, squirrels and woodchucks and these
EP

models have provided crucial insights in HBV virology and subsequent advances in
C

antiviral therapy (5).

AC

HBV is a pararetrovirus. It is a DNA virus but its replication strategy is reminiscent of

RNA retro-viruses, as it replicates by reverse transcription via an RNA intermediate.

One of the salient features of the HBV viral transcriptase is that it lacks proof reading,

which renders HBV highly susceptible to acquiring mutations during each cycle of

replication especially G-A hyper-mutation. This gives rise to various genotypes and

mutations that usually confer either a survival advantage or an escape mechanism to

3
 ACCEPTED MANUSCRIPT
the virus and they have significant clinical implications to the natural history and the

disease outcome of HBV.

Another unique feature of HBV is its ability to form a minichromosome during viral

replication, which acts as a template for its RNA transcripts. This is also known as the

PT
cccDNA transcriptional template and it can persist in the hepatocyte indefinitely

ensuring chronic lifelong viral persistence until the host cell is eliminated. This poses

RI
a significant problem for the treatment of HBV. To date, current antivirals can merely

SC
suppress viral replication but cannot eliminate it altogether from the liver. HBV, like

most retroviruses, has oncogenic potential. In fact it is the leading cause of

U
hepatocellular carcinoma, the third leading cause of cancer deaths worldwide.
AN
The virology of HBV is complex and not entirely understood. However a thorough
M

insight in the virological aspects of HBV provides a deeper understanding of the

disease phenotype and aid in the discovery of novel therapeutics. In this article, we
D

review the key virological aspects of HBV with a focus on clinical and pathological
TE

implications.
C EP
AC

4
 ACCEPTED MANUSCRIPT
Structure of the Virus

HBV virions were originally named Dane particles after the researcher who led the

team that first succeeded in identifying the HBV via electron microscopy (6). The

virion is 48-52 nm in diameter, consisting of a nucleocapsid core enclosed in a

PT
glycolipid envelope (7, 8).

RI
The envelope is made up of lipids and three surface proteins. The core is an

icosahedral nucleocapsid of 30-32 nm in diameter typically made up of either 180 or

SC
240 core subunits with triangulation numbers (T) 3 or 4, respectively. Capsids with

the T=4 configuration appear to be the predominant ones detected in active infection

U
(9, 10). Cryoelectron microscopy studies have revealed that core particles form spikes
AN
on the surface of the particle and these spike tips have strongly immunogenic
M

epitopes. The core contains the genome from which all viral proteins are transcribed.
D

Subviral particles
TE

In natural infections with HBV, over and above the production of fully formed

virions, subviral particles (SVP) are also formed and secreted in 1,000- 100, 000 fold
EP

excess of virions(11). SVP consist of surface proteins and can be either of spherical or
C

filamentous conformation; spheres are 20-25 nm in diameter and filaments are 22 nm

AC

in diameter and are of variable length(12). SVP are made up of the three HBV

envelope proteins that are collectively known as the surface antigen (HBsAg) (6, 13).

SVP do not contain any viral DNA and therefore they are not infectious (14) but they

have immunogenic properties. In fact, they were used in early vaccine development

(15) and it is thought that they are secreted in such excess in order to neutralize anti-

HBs antibodies hence increasing immune tolerance to promote viral persistence (16).

5
 ACCEPTED MANUSCRIPT
SVP have also been shown to enhance viral replication and gene expression and

therefore, despite not being directly infectious themselves, they increase infectivity

(17, 18).

PT
RI
U SC
AN
M
D
TE
C EP
AC

6
 ACCEPTED MANUSCRIPT
The genome

The HBV genome is a partially double-stranded DNA strand of approximately 3200

base pairs (dependent on the genotype), in a relaxed circular conformation (rcDNA)

(19). The strands overlap at the 5’ end and the minus strand is complete but the plus

PT
strand has a gap of about 600 nucleotides and the position of the 3’ end is variable

RI
(20). HBV has an endogenous polymerase to incorporate nucleotides in order to

complete the genome forming a full double strand during viral replication (20-22).

SC
The fully circularized HBV genome is made up of four overlapping and frame-shifted

U
reading frames (ORFs) which are Pol (P), Core (C), Surface (S) and X and they
AN
encode for the seven HBV proteins. They also encode for four promoter regions that
M

help initiate transcription and two enhancers that promote gene transcription when

bound by transcription activators. The genome also has cis- regulating elements
D

(CRE), which are regions of noncoding DNA that regulate transcription of nearby
TE

genes usually by acting as binding sites for transcription factors.

The overlapping ORF have significant implications in the setting of mutagenesis

EP

as a mutation in one reading frame can affect the overlapping frame and its
C

associated protein.
AC

Pol is the longest reading frame covering 70% of the viral genome and encodes

the viral polymerase. HBV polymerase includes four functional sub-domains that

are enzymes required for DNA synthesis: the reverse transcriptase required for

synthesis of the plus strand, a carboxy terminal region that has ribonuclease H

activity, a spacer region as well as a terminal domain which primes the

pregenomic (pg)RNA for the synthesis of the minus strand of DNA.

7
 ACCEPTED MANUSCRIPT
The S- ORF encodes for three HBV surface proteins: large (LHBs), middle (MHBs),

and small (SHBs) protein but the proteins are translated from two different mRNAs:

LHBs is translated from a long 2.4 kb transcript whereas MHBs and SHBs are

translated from a slightly shorter 2.1 kb transcript. All three envelope proteins

terminate at the same 3’ end but their starting 5’ end is staggered such that L and M

PT
proteins have extra domains, the Pre-S1 and pre-S2 respectively.

RI
SHBs is the smallest of the three proteins, made up to 226 amino acids and is

synthesised in the endoplasmic reticulum (ER). An unusual feature of HBV envelope

SC
proteins in that they have multiple transmembrane domains that span the ER with

loops of amino acids internal and external to the cytosol.

U
SHBs spans the ER membrane via four transmembrane domains (TM 1-4) which are
AN
linked by internal and external loops (23). The loop of amino acids linking TM2 and 3
M

is external to the ER and made of amino acids 99–161. This loop is known as the ‘a’

determinant and it is of particular virological and clinical significance as it is a major

D

antigenic determinant of HBV as detailed below.

TE

The exact role of the MHBs remains unclear as it is not required for either viral or

subviral particle secretion nor for infectivity (12).

EP

In contrast, LHBs is an important protein in the viral life cycle, essential for viral
C

entry. The PreS1 domain of the L protein has a 75 amino acid domain at the N
AC

terminal that requires myristylation that is essential for viral infectivity (24, 25).

LHBs also has an important role in encapsidation of the core during viral replication

as the mature virion is formed and secreted.

The precore/core ORF encode for the core protein as well as the precore antigen,

HBeAg. Full translation of the core ORF results in the formation of HBeAg, which is

secreted in great excess of virions (26). HBeAg is the only protein to be modified

8
 ACCEPTED MANUSCRIPT
post-translationally and prior to being secreted, it is proteolytically processed at both

C and N termini.

HBeAg is an accessory protein that is not required for structural stability, viral

replication, secretion or infectivity (27). HBeAg is a serological marker for active

HBV infection and replication and is regarded as necessary for the establishment of

PT
persistence. Its exact role is unknown but it is thought to enable immune escape by

RI
acting as an immunomodulator and tolerogen (28). The exact mechanisms are unclear,

whether it is by suppressing the immune response to the virus (29, 30) or by acting as

SC
a decoy particle for the immune system (31).

The X ORF encodes for HBx, the X protein, which is not a structural protein but

U
is required for cccDNA transcription and is therefore a key regulator of HBV
AN
replication. It also confers a carcinogenic potential to HBV (32, 33).
M

The HBV life cycle

D

The life cycle of HBV is well characterized and can be classified in 3 stages: early,
TE

middle, and late (34). The main events are depicted in Figure 1. The early stages

include the processes of viral attachment and entry into the hepatocyte and then the
EP

generation and transcription of the viral minichromosome. HBV cellular entry has

only recently been elucidated. HBV initially binds, in the space of Disse in the liver,
C

to heparan sulfate proteoglycans (35) expressed on the surface of hepatocytes, then

AC

with high-specificity to the sodium taurocholate cotransporting polypeptide (NTCP)

receptor via the preS1 attachment site and subsequently becomes internalised in

endosomal vesicles (36, 37).

After entry into the hepatocyte, surface proteins are removed and the nucleocapsid is

actively transported to the nucleus via microtubules, where it is uncoated and the

9
 ACCEPTED MANUSCRIPT
genome is delivered to the nucleus (38). The small diameter of the capsid allows it to

pass through the nuclear pores. At this point in the viral life cycle, the genome is still

in its usual relaxed circular conformation with an incomplete plus strand.

HBV is a pararetrovirus by virtue of its replication strategy: It is a DNA virus but it

replicates via reverse transcription using an RNA intermediate (39) (5, 40). However,

PT
it differs from retroviruses in distinct ways. Firstly, in HBV replication, each protein

RI
is translated from its own mRNA except for Pol and Core which are both translated

from the same mRNA, the pgRNA. Secondly, proteins are not processed post-

SC
translationally except for the precore protein, which undergoes proteolysis. Finally,

unlike retroviruses, HBV DNA is first converted to a mini-chromosome where the

U
viral DNA can be found as a covalently closed circular DNA (cccDNA) molecule,
AN
which is the functional equivalent of an intracellular provirus. To form cccDNA, rc-
M

DNA is first converted to a fully double stranded genome. The rc-DNA has a

complete minus strand but a gap in the plus strand, the 5’ end of the minus strand is
D

linked to the P protein whilst the 5’ end of the plus strand contains a primer for plus
TE

strand synthesis. cccDNA binds to histones and other chromatinizing proteins to form

a mini chromosome that serves as the template for transcription of HBV mRNA (41,
EP

42).
C
AC

cccDNA

cccDNA is an important feature of HBV infection that determines multiple significant

clinical characteristics including chronicity and lifelong persistence, carcinogenesis

and relative inefficacy of antiviral treatment.

cccDNA is a stable form of viral DNA that allows HBV to persist in the hepatocyte

hence ensuring the ongoing production of virus. cccDNA has yet to be identified in

10
 ACCEPTED MANUSCRIPT
non-authentic host cells, such as transgenic animals, suggesting that specific host

factors are required for its formation, which contributes to the species specificity of

HBV. cccDNA is produced early, within 24 hours (43), and in large quantities. In a

natural HBV infection there are more than 50 copies of cccDNA in a hepatocyte due

to intracellular amplification (44-46) from pgRNA reverse transcription or from

PT
multiple rounds of virion reinfection. It can persist in the infected hepatocyte for up to

RI
60 days and is not lost with cell division.

Unlike other hepatotropic viruses, HBV DNA can integrate into the host genome.

SC
A common site of viral integration is near the gene for human telomerase reverse

transcriptase (hTERT). This is not necessary for replication but it ensures

U
persistence in the hepatocyte. Integration also confers oncogenic potential to the
AN
virus as it can interfere with cellular signaling, proliferation and apoptosis.
M

cccDNA is not susceptible to eradication with current antiviral therapy (11, 44, 47).
D

Current antiviral therapy can efficiently suppress viral replication, but permanent
TE

eradication of HBV is virtually impossible to achieve due to the cccDNA reservoir.

cccDNA elimination in chronic infection remains the holy grail of HBV therapeutics.
EP

The middle stages in the viral life cycle include the transcription of the viral proteins.
C

From the cccDNA minichromosome template, five RNA species of different sizes are
AC

transcribed by the host cell RNA polymerase II. The cccDNA also encodes for four

promoters and two enhancers. The enhancers have binding sites for transcription

factors, which are liver specific, HNF3 and HNF4, yet another mechanism for the

virus to maintain hepatotropism (48).

There are two genomic transcripts of 3.5kb each and three sub-genomic transcripts of

2.4 kb, 2.1 kb and 0.7 kb. The subgenomic 2.4 kb transcript serves as mRNA for the

11
 ACCEPTED MANUSCRIPT
translation of the pre-S1 protein whilst the 2.1 kb is the mRNA for the translation of

both Pre-S2, and S and the 0.7 kb transcript functions as mRNA for the translation of

the X protein (49). The 5’ end of the 2.1 kb mRNA is heterogeneous and the Pre-S2

start codon is only present in a proportion of transcripts such that the S protein is far

more abundant than pre-S2.

PT
The 3.5 kb genomic transcripts include the precore mRNA and pregenomic RNA

RI
(pgRNA). The precore mRNA is similar to the core protein but has an extension at the

N terminal end and it encodes for HBeAg, and as such is the first HBV protein

SC
synthesized.

PgRNA encodes for HBcAg and Pol. PgRNA is also the template for reverse

U
transcription to generate HBV DNA. pgRNA is converted to double stranded DNA
AN
(ds-DNA) by reverse transcription. Reverse transcription has been well reviewed in
M

the literature and will not be reprised in great detail here (50). DNA synthesis starts

when the viral polymerase binds to an encapsidation signal of the pgRNA. This
D

initiates encapdisation to form new nucleocapsids (51) (52-54). Reverse transcription

TE

of the RNA occurs inside the new nucleocapsid to form rc-DNA. A short DNA

sequence is generated using a region of the epsilon loop as template (55-57). The
EP

template then switches to the 3’ end and the minus strand synthesis is initiated. The
C

plus strand is then synthesized and the genome is circularized.

AC

The newly synthesized DNA then either get recycled to the nucleus to maintain the

cccDNA pool or they are packaged with surface proteins and exported as new virions.

Once infected patients will harbour cccDNA and replication intermediates such as

pregenomic RNA (pgRNA) in the hepatocyte indefinitely until the hepatocyte is

eliminated (58). Even after seroclearance of HBV, with the formation of antibodies to

the surface antigen (anti-HBs), a risk of reactivation is still present in the context of

12
 ACCEPTED MANUSCRIPT
immunosuppression (58-60). HBV DNA also has the ability to integrate in the host

genome. Double stranded linear (dsl) DNA is formed as a by-product of viral

replication and dsl DNA can integrate via a process of recombination in the host

chromosome.

Splicing of the pgRNA can also occur with major deletions in the mRNA, producing

PT
variants. Splice variants are considered defective but they have important clinical

RI
implications (see splice variants later in the text).

SC
The late events of the viral life cycle include viral assembly and release. In the

cytosol, mature rc-DNA is assembled into new nucleocapsids and they are enveloped

U
prior to being exported from the hepatocyte. Virions will only be enveloped once the
AN
genome is fully formed and mature with a minus DNA strand and a partial plus strand
M

(40) although empty capsids have been detected.

For the formation of new virions, capsids move towards intracellular membranes
D

containing the three envelope proteins. These intracellular membranes act as budding
TE

sites for the formation of new virions. Surface proteins are essential for the final

stages of viral assembly and the budding of new HBV capsids. The S protein consists
EP

of a luminal N-terminal sequence, two transmembrane regions with a cysolic loop in

C

between and a luminal domain that includes the ‘a’ determinant also including the
AC

glycosylation site (61) at residue 146. PreS1 has a dual topology and in the virion half

of PreS1 ends up on the internal surface of the virion, which is required for the

envelopment of core particles and the other half of PreS1 is expressed externally as it

is required for subsequent viral attachment (61) on release. Following envelopment,

the virions are deposited in the lumen of the ER and released.

13
 ACCEPTED MANUSCRIPT
Virions rely on the multivesicular body (MVB) pathway that generates intraluminal

vesicles (62) in order to be released from the cell. The MVB pathway depends on the

endosomal sorting complex required for transport (ESCRT) system that comprises the

ESCRT-0, -I, -II, and –III complexes and associated proteins. The activation of

ESCRT-III in particular brings about budding and membrane fission allowing the

PT
virion to be released out of the hepatocyte.

RI
SVPs are formed independent of capsids as the envelope proteins for oligomers that

bud off as spheres or filaments (63). They do not depend on the ESCRT complex for

SC
release from the cell (64), but are processed via the ER-Golgi pathway.

Splicing
U
AN
Splice donor (SD) and acceptor (SA) sites can be detected throughout the pgRNA.
M

Thus, splicing can also occur in the pgRNA that involves deletions of nucleotides at

specific sites. Spliced transcripts were first identified in hepatoma cell lines
D

containing a deletion of 1223 bases in the core mRNA. Despite that deletion, a core
TE

protein was still transcribed but the function of those spliced transcripts was

unknown. The junction of the deleted sequences revealed splice donor and acceptor
EP

sequences that appear to be relatively well conserved and the splice transcript does
C

not seem to be viable when there is a mutation at the receptor site (65). All spliced
AC

RNA lack a functional pol ORF. Spliced RNA transcripts have been isolated in

hepatoma cell lines (66). Reverse transcription of spliced RNA results in the

formation of splice variants which are defective viruses (67).

At least five major splice donor and acceptor sites have been identified (68, 69) and

the number of possible combinations is high. However, splice variants appear to

14
 ACCEPTED MANUSCRIPT
favour particular genotypes. For instance the most common splice variant, Splice

Variant 1 (SP1), has been reported in genotypes A,C,D and E (70).

In eukaryote cells, splicing is an almost universal process for all pre-mRNA in order

to remove introns prior to export out of the nucleus for translation. In contrast, in

RNA viruses and in HBV the unspliced RNA is the main RNA that is to be packaged

PT
and exported. This is because in HBV, there is a post-transcriptional regulatory

RI
element (PRE), at nt 1217–1582 which facilitates the export of RNA without prior

splicing (71).

SC
Splice variants are not routinely tested in the clinical setting but they are common,

with some case series reporting an incidence of up to 96% in serum samples of

patients with chronic HBV (72).

U
AN
Spliced pgRNA can form a novel protein called the HBV splice-generated protein
M

(HBSP) (73). HBSP is associated with increased viral replication as manifested by an

increased expression of core protein as well as secretion of HBeAg (72). Splice

D

variants have also been associated with an increased progression to chronicity

TE

following acute infection with HBV (72), higher rates of liver cirrhosis and

hepatocellular carcinoma (73, 74).

C EP

Genotypes
AC

The reverse transcriptase in HBV replication lacks proof-reading, which makes HBV

highly susceptible to acquiring mutations during each cycle of viral replication. The

mutation rate for HBV is estimated at 104- 106 nucleotide substitutions/ site/ year,

about 100 times more than that of any other DNA virus (75). The high mutagenesis

rate accounts for the formation of genotypes and viral variants. HBV genotype

classification is based on phylogenetic analysis of the entire genome. They differ from

15
 ACCEPTED MANUSCRIPT
one other by a sequence variation of 8% or more across the entire genome (76) or by

4% or more in the S reading frame (77). There are 10 genotypes of HBV namely

genotypes A-J. Genotypes can be further categorised into sub-genotypes that differ

from one another by a sequence divergence of 4-8% across the genome. The

geographical distribution of HBV genotypes is well characterized. Genotypes B and C

PT
is prevalent in Asia, genotype E is found in sub-Saharan Africa and Genotypes A and

RI
D are prevalent in Europe. Sub-genotypes also have distinct geographical

distributions that appear to evolve independently. For instance, sub-genotype C4 is

SC
found exclusively in the Northern Territory of Australia (78), sub-genotype A1 (Aa)

is predominantly found in Africa whilst A2 (Ae) is prevalent in Europe and North

U
America (79, 80). Similarly sub-genotype B1 (Bj) is almost exclusively found in
AN
Japan while B2 (Ba) is prevalent throughout the rest of Asia.
M

There is good epidemiological evidence that genomic variability of HBV is associated

with different disease outcomes. However most epidemiological data regarding

D

disease phenotype are comparative in nature. Most studies have been done in Asia
TE

and therefore are a comparison of genotypes B and C. Similarly studies done in

Europe are usually a comparison of the two most prevalent genotypes, namely A and
EP

D.
C

Genotype C has been associated with more severe disease outcomes compared to
AC

genotype B. Similarly, genotype D confers worse prognosis than genotype A. The

clinical outcomes examined in most epidemiological studies include progression to

chronicity (81-83), time to HBeAg seroconversion (84-89), histologic activity,

cirrhosis (85), hepatocellular carcinoma and response to interferon treatment (90).

Genotypes are also associated with specific mutations including basal core promoter

16
 ACCEPTED MANUSCRIPT
(BCP) and precore (PC) mutations that are associated with more severe disease

phenotypes.

Mutations

PT
Point mutations also commonly arise during viral replication in context of reverse

transcription and the G to A hyper-mutation. The variation is in keeping with the

RI
Darwinian concept of survival of the fittest, where variants that can survive

SC
endogenous and exogenous selection pressures will have a survival advantage. In the

clinical setting, variants commonly arise during the immune active phase of disease

U
when immune pressures are high or as a result of drug exposure. Due to the
AN
overlapping nature of the ORFs, a mutation in one frame can affect the function of the

protein encoded by that frame but also the protein encoded by the overlapping reading
M

frame.
D
TE

BCP and PC mutations

The most common mutations detected in HBV are the PC and BCP mutations. These
EP

are associated with reduced or abolished HBeAg production respectively, and they
C

emerge late in the natural history of chronic hepatitis B (CHB), during the immune
AC

reactive phase of HBeAg seroconversion (91).

BCP and PC mutations have been associated with advanced fibrosis and cirrhosis and

an increased risk of HCC (92-96). They have also been associated with a higher

number of disease flares and necro-inflammation (97, 98), fulminant liver failure and

cirrhosis-related complications such as decompensation (99). The presence of BCP

17
 ACCEPTED MANUSCRIPT
and PC mutations is also associated with a poor response to interferon therapy (100)

and a lower rate of HBsAg loss with tenofovir therapy (101).

PreS/S mutations

Development of substitutions at the ‘a’ determinant affect the antigenicity of HBsAg.

PT
This has significant clinical and public health implications as it can lead to vaccine

RI
escape and failure of immunoglobulin treatment as well as false negative results by

the commercially used HBsAg assays (102, 103). The most commonly reported

SC
vaccine resistance mutation associated with vaccine failure is the G145R, which

reduces anti-HBs binding capacity hence allowing vaccine escape (104). The

U
mutation has also been associated with fulminant flares of HBV (105). Although
AN
vaccine-resistant variants appear to emerge more commonly in areas of universal
M

vaccination, they emerge so slowly that they should not impact the global

immunisation recommendation (106). S mutations can also arise as result of

D

mutations in the P ORF that are generally caused by exposure to antivirals, a

TE

phenomenon commonly called antiviral-drug-associated S gene mutations (107). The

S mutations are not only associated with a change in protein antigenicity but can also
EP

be associated with increased oncogenic potential. For instance, the rtA181T that arises
C

in the P gene can cause the formation of a stop codon at position 172 in the S protein,
AC

the sW172* (stop codon) mutation, which is associated with an increased risk for

developing hepatocellular carcinoma (108) in vivo.

Occult HBV infection is increasingly being reported, whereby HBsAg in the serum is

reported as negative (below lower limit of detection, LLOD) by conventional assays

but there is HBV DNA present in the liver. The inability for the assay to detect

HBsAg may be due to very low levels of HBsAg in serum or may also be due to the

18
 ACCEPTED MANUSCRIPT
presence of a modified HBsAg that the assay fails to detect despite being present at

high levels. The PreS gene is also prone to mutations. The PreS region is strongly

immunogenic. The PreS deletion mutant is common in genotype C (109) and it may

impair the secretion of HBsAg hence increasing the intracellular stress of the

hepatocyte which is associated with oncogenicity (110-113).

PT
RI
Pol variants

The YMDD (tyrosine, methionine, aspartate, aspartate) motif is preserved across all

SC
genotypes and it is essential for active viral replication. Currently, the mainstay of

antiviral treatment is the use of nucleot(s)ide analogues (NA) that directly target the

U
reverse transcriptase. Changes in the YMDD motif arise as a result of drug pressures
AN
and are associated with antiviral resistance. Antiviral resistance mutations often also
M

compromise viral replication and as a result, compensatory mutations develop.

Development of resistance to antivirals typically manifests clinically as a

D

breakthrough in viral titres or a rise in biochemical markers in particular serum

TE

aminotransferases and can result in clinical deterioration of the patient (49).

Lamivudine was one of the first antiviral agents used in the treatment of HBV but the
EP

rate of resistance is high, in the order of 70% after 48 months of treatment (114).
C

Adefovir also has a high resistance rate of approximately 30% after 5 years of
AC

treatment (115). Lamivudine and adefovir are now not recommended at all whilst

entecavir and tenofovir are first line therapy due to their high genetic barrier and

potency.

Of the two agents, entecavir has the lesser favorable resistance profile with a rate of

roughly 1.2% after 5 years in treatment naïve patients (116). For entecavir resistance

to occur at least three mutations must be present: rtL180M, rtM204V and either

19
 ACCEPTED MANUSCRIPT
rtT184G/S or rtS202I/G or rtM250V (117). To date, there is no resistance reported for

tenofovir. The rtA194T mutations, which confers resistance to adefovir was reported

to cause partial tenofovir resistance but in clinical practice, virological or biochemical

breakthrough has not been observed with Tenofovir (118).

PT
X mutants

RI
The X protein is a regulatory protein and is a key protein in viral persistence. It is also

important in carcinogenesis. X gene mutants have been associated in HCC (119).

SC
Integrated X gene sequences are commonly seen in HCC tissue (120). Because of the

gene overlap in HBV, BCP variants (A1762T, G1764A) result in codon changes in X

at x130 and x131(119).

U
AN
M

Summary/ Practice points

• HBV is a hepadnavirus and it is species-specific and hepatotropic.

D

• The virus is made up of a nucleocapsid core which contains the genome

TE

enclosed by an envelope composed of lipids and surface proteins.

• HBV is a DNA virus and encodes seven main proteins namely polymerase,
EP

core, precore, X proteins and 3 surface envelope proteins.

•
C

Despite being a DNA virus, HBV replicates like an RNA virus via an
AC

intermediate known as pgRNA. It forms a minichromosome cccDNA that can

ensure viral persistence and from which all viral mRNAs are transcribed.

• Genotypes and variants are common in HBV due to a lack of proof-reading in

the reverse transcriptase. These are associated with major differences in

disease phenotype.

20
 ACCEPTED MANUSCRIPT
• Mutations in the precore antigen are associated with a more aggressive disease

phenotype, mutation in Pol are associated with resistance to antivirals,

mutations in S are associated with vaccine resistance and occult HBV and X

mutations are associated with a high rate of liver cancer.

Research Agenda:

PT
• The virology of HBV is complex and not entirely elucidated, making it an

RI
elusive virus to treat and eradicate.

• The exact role of HBeAg is not entirely clear and detailed virological and

SC
immunological studies are required

• Further virological and immunological studies in the role of the X protein are

U
necessary to assess its carcinogenic potential
AN
• Clinical and animal studies are required to asses the immunology of common
M

and emerging mutations

• Detailed virological studies are required to elucidate the biology of cccDNA

D

with an aim to develop novel therapeutics

TE

Conflict of interest:

Stephen Locarnini has received consultant fees as well as grant support from Gilead
EP

Sciences Inc, Arrowhead Research Corp and Spring Bank Pharmaceuticals Inc.
C

Zina Valaydon has no conflict of interest.

AC

21
 ACCEPTED MANUSCRIPT
References
1. <ICTV 5th Report.pdf>.https://talk.ictvonline.org/ictv-
reports/ictv_online_report
2. Bancroft WH, Snitbhan R, Scott RM, Tingpalapong M, Watson WT,
Tanticharoenyos P, et al. Transmission of hepatitis B virus to gibbons by exposure to
human saliva containing hepatitis B surface antigen. The Journal of infectious
diseases. 1977;135(1):79-85.
3. Barker LF, Chisari FV, McGrath PP, Dalgard DW, Kirschstein RL, Almeida
JD, et al. Transmission of type B viral hepatitis to chimpanzees. The Journal of

PT
infectious diseases. 1973;127(6):648-62.
4. Dupinay T, Gheit T, Roques P, Cova L, Chevallier-Queyron P, Tasahsu SI, et
al. Discovery of naturally occurring transmissible chronic hepatitis B virus infection

RI
among Macaca fascicularis from Mauritius Island. Hepatology. 2013;58(5):1610-20.
5. Mason WS, Aldrich C, Summers J, Taylor JM. Asymmetric replication of
duck hepatitis B virus DNA in liver cells: Free minus-strand DNA. Proceedings of the

SC
National Academy of Sciences of the United States of America. 1982;79(13):3997-
4001.
6. Dane DS, Cameron CH, Briggs M. Virus-like particles in serum of patients
with Australia-antigen-associated hepatitis. Lancet. 1970;1(7649):695-8.

U
7. Dryden KA, Wieland SF, Whitten-Bauer C, Gerin JL, Chisari FV, Yeager M.
Native hepatitis B virions and capsids visualized by electron cryomicroscopy.
AN
Molecular cell. 2006;22(6):843-50.
8. Seitz S, Urban S, Antoni C, Bottcher B. Cryo-electron microscopy of hepatitis
B virions reveals variability in envelope capsid interactions. The EMBO journal.
M

2007;26(18):4160-7.
9. Glebe D, Bremer CM. The molecular virology of hepatitis B virus. Seminars
in liver disease. 2013;33(2):103-12.
D

10. Roseman AM, Berriman JA, Wynne SA, Butler PJ, Crowther RA. A structural
model for maturation of the hepatitis B virus core. Proceedings of the National
Academy of Sciences of the United States of America. 2005;102(44):15821-6.
TE

11. Ganem D, Prince AM. Hepatitis B virus infection--natural history and clinical
consequences. The New England journal of medicine. 2004;350(11):1118-29.
12. Glebe D, Urban S. Viral and cellular determinants involved in hepadnaviral
EP

entry. World journal of gastroenterology : WJG. 2007;13(1):22-38.

13. Bayer ME, Blumberg BS, Werner B. Particles associated with Australia
antigen in the sera of patients with leukaemia, Down's Syndrome and hepatitis.
C

Nature. 1968;218(5146):1057-9.
14. Schadler S, Hildt E. HBV life cycle: entry and morphogenesis. Viruses.
AC

2009;1(2):185-209.
15. Blumberg BS. Hepatitis B virus and the control of hepatocellular carcinoma.
IARC Sci Publ. 1984(63):243-61.
16. Ganem D. Assembly of hepadnaviral virions and subviral particles. Current
topics in microbiology and immunology. 1991;168:61-83.
17. Bruns M, Miska S, Chassot S, Will H. Enhancement of hepatitis B virus
infection by noninfectious subviral particles. Journal of virology. 1998;72(2):1462-8.
18. Seeger, C., F. Zoulim, and W. S. Mason. 2007. Hepadnaviruses, p. 2977–
3030. In D. M. Knipe (ed.), Fields virology, 5th ed. Lippincott Williams & Wilkins,
Philadelphia, PA.

22
 ACCEPTED MANUSCRIPT
19. Landers TA, Greenberg HB, Robinson WS. Structure of hepatitis B Dane
particle DNA and nature of the endogenous DNA polymerase reaction. Journal of
virology. 1977;23(2):368-76.
20. Summers J, O'Connell A, Millman I. Genome of hepatitis B virus: restriction
enzyme cleavage and structure of DNA extracted from Dane particles. Proceedings of
the National Academy of Sciences of the United States of America.
1975;72(11):4597-601.
21. Kaplan PM, Greenman RL, Gerin JL, Purcell RH, Robinson WS. DNA
polymerase associated with human hepatitis B antigen. Journal of virology.

PT
1973;12(5):995-1005.
22. Robinson WS, Greenman RL. DNA polymerase in the core of the human
hepatitis B virus candidate. Journal of virology. 1974;13(6):1231-6.

RI
23. Berting A, Hahnen J, Kroger M, Gerlich WH. Computer-aided studies on the
spatial structure of the small hepatitis B surface protein. Intervirology. 1995;38(1-
2):8-15.

SC
24. Macrae DR, Bruss V, Ganem D. Myristylation of a duck hepatitis B virus
envelope protein is essential for infectivity but not for virus assembly. Virology.
1991;181(1):359-63.
25. Gripon P, Le Seyec J, Rumin S, Guguen-Guillouzo C. Myristylation of the

U
hepatitis B virus large surface protein is essential for viral infectivity. Virology.
1995;213(2):292-9.
AN
26. Garcia PD, Ou JH, Rutter WJ, Walter P. Targeting of the hepatitis B virus
precore protein to the endoplasmic reticulum membrane: after signal peptide cleavage
translocation can be aborted and the product released into the cytoplasm. The Journal
M

of cell biology. 1988;106(4):1093-104.

27. Schlicht HJ, Salfeld J, Schaller H. The duck hepatitis B virus pre-C region
encodes a signal sequence which is essential for synthesis and secretion of processed
D

core proteins but not for virus formation. Journal of virology. 1987;61(12):3701-9.
28. Chen M, Sallberg M, Hughes J, Jones J, Guidotti LG, Chisari FV, et al.
Immune tolerance split between hepatitis B virus precore and core proteins. Journal of
TE

virology. 2005;79(5):3016-27.
29. Riordan SM, Skinner N, Kurtovic J, Locarnini S, Visvanathan K. Reduced
expression of toll-like receptor 2 on peripheral monocytes in patients with chronic
EP

hepatitis B. Clinical and vaccine immunology : CVI. 2006;13(8):972-4.

30. Visvanathan K, Skinner NA, Thompson AJ, Riordan SM, Sozzi V, Edwards
R, et al. Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the
C

precore protein. Hepatology. 2007;45(1):102-10.

31. Kimura T, Ohno N, Terada N, Rokuhara A, Matsumoto A, Yagi S, et al.
AC

Hepatitis B virus DNA-negative dane particles lack core protein but contain a 22-kDa
precore protein without C-terminal arginine-rich domain. The Journal of biological
chemistry. 2005;280(23):21713-9.
32. Lucifora J, Arzberger S, Durantel D, Belloni L, Strubin M, Levrero M, et al.
Hepatitis B virus X protein is essential to initiate and maintain virus replication after
infection. Journal of hepatology. 2011;55(5):996-1003.
33. Wei Y, Neuveut C, Tiollais P, Buendia MA. Molecular biology of the
hepatitis B virus and role of the X gene. Pathologie-biologie. 2010;58(4):267-72.
34. Locarnini S. Molecular virology and the development of resistant mutants:
implications for therapy. Seminars in liver disease. 2005;25 Suppl 1:9-19.
35. Leistner CM, Gruen-Bernhard S, Glebe D. Role of glycosaminoglycans for
binding and infection of hepatitis B virus. Cellular microbiology. 2008;10(1):122-33.

23
 ACCEPTED MANUSCRIPT
36. Yan H, Zhong G, Xu G, He W, Jing Z, Gao Z, et al. Sodium taurocholate
cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.
eLife. 2012;1.
37. Ni Y, Lempp FA, Mehrle S, Nkongolo S, Kaufman C, Falth M, et al. Hepatitis
B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-
specific entry into hepatocytes. Gastroenterology. 2014;146(4):1070-83.
38. Rabe B, Vlachou A, Pante N, Helenius A, Kann M. Nuclear import of
hepatitis B virus capsids and release of the viral genome. Proceedings of the National
Academy of Sciences of the United States of America. 2003;100(17):9849-54.

PT
39. Seeger C, Hu J. Why are hepadnaviruses DNA and not RNA viruses? Trends
in microbiology. 1997;5(11):447-50.
40. Summers J, Mason WS. Replication of the genome of a hepatitis B--like virus

RI
by reverse transcription of an RNA intermediate. Cell. 1982;29(2):403-15.
41. Newbold JE, Xin H, Tencza M, Sherman G, Dean J, Bowden S, et al. The
covalently closed duplex form of the hepadnavirus genome exists in situ as a

SC
heterogeneous population of viral minichromosomes. Journal of virology.
1995;69(6):3350-7.
42. Bock CT, Schranz P, Schroder CH, Zentgraf H. Hepatitis B virus genome is
organized into nucleosomes in the nucleus of the infected cell. Virus genes.

U
1994;8(3):215-29.
43. Tagawa M, Omata M, Okuda K. Appearance of viral RNA transcripts in the
AN
early stage of duck hepatitis B virus infection. Virology. 1986;152(2):477-82.
44. Zhu Y, Yamamoto T, Cullen J, Saputelli J, Aldrich CE, Miller DS, et al.
Kinetics of hepadnavirus loss from the liver during inhibition of viral DNA synthesis.
M

Journal of virology. 2001;75(1):311-22.

45. Tuttleman JS, Pourcel C, Summers J. Formation of the pool of covalently
closed circular viral DNA in hepadnavirus-infected cells. Cell. 1986;47(3):451-60.
D

46. Wu TT, Coates L, Aldrich CE, Summers J, Mason WS. In hepatocytes

infected with duck hepatitis B virus, the template for viral RNA synthesis is amplified
by an intracellular pathway. Virology. 1990;175(1):255-61.
TE

47. Addison WR, Walters KA, Wong WW, Wilson JS, Madej D, Jewell LD, et al.
Half-life of the duck hepatitis B virus covalently closed circular DNA pool in vivo
following inhibition of viral replication. Journal of virology. 2002;76(12):6356-63.
EP

48. Moolla N, Kew M, Arbuthnot P. Regulatory elements of hepatitis B virus

transcription. Journal of viral hepatitis. 2002;9(5):323-31.
49. Locarnini S, Omata M. Molecular virology of hepatitis B virus and the
C

development of antiviral drug resistance. Liver International. 2006;26(S2):11-22.

50. Nassal M. Hepatitis B viruses: reverse transcription a different way. Virus
AC

research. 2008;134(1-2):235-49.
51. Bartenschlager R, Schaller H. Hepadnaviral assembly is initiated by
polymerase binding to the encapsidation signal in the viral RNA genome. The EMBO
journal. 1992;11(9):3413-20.
52. Junker-Niepmann M, Bartenschlager R, Schaller H. A short cis-acting
sequence is required for hepatitis B virus pregenome encapsidation and sufficient for
packaging of foreign RNA. The EMBO journal. 1990;9(10):3389-96.
53. Knaus T, Nassal M. The encapsidation signal on the hepatitis B virus RNA
pregenome forms a stem-loop structure that is critical for its function. Nucleic Acids
Res. 1993;21(17):3967-75.
54. Pollack JR, Ganem D. An RNA stem-loop structure directs hepatitis B virus
genomic RNA encapsidation. Journal of virology. 1993;67(6):3254-63.

24
 ACCEPTED MANUSCRIPT
55. Wang GH, Seeger C. The reverse transcriptase of hepatitis B virus acts as a
protein primer for viral DNA synthesis. Cell. 1992;71(4):663-70.
56. Weber M, Bronsema V, Bartos H, Bosserhoff A, Bartenschlager R, Schaller
H. Hepadnavirus P protein utilizes a tyrosine residue in the TP domain to prime
reverse transcription. Journal of virology. 1994;68(5):2994-9.
57. Zoulim F, Seeger C. Reverse transcription in hepatitis B viruses is primed by a
tyrosine residue of the polymerase. Journal of virology. 1994;68(1):6-13.
58. Vierling JM. The immunology of hepatitis B. Clinics in liver disease.
2007;11(4):727-59, vii-viii.

PT
59. Chang JJ, Lewin SR. Immunopathogenesis of hepatitis B virus infection.
Immunology and cell biology. 2007;85(1):16-23.
60. Rehermann B, Ferrari C, Pasquinelli C, Chisari FV. The hepatitis B virus

RI
persists for decades after patients' recovery from acute viral hepatitis despite active
maintenance of a cytotoxic T-lymphocyte response. Nat Med. 1996;2(10):1104-8.
61. Will H, Reiser W, Weimer T, Pfaff E, Buscher M, Sprengel R, et al.

SC
Replication strategy of human hepatitis B virus. Journal of virology. 1987;61(3):904-
11.
62. Watanabe T, Sorensen EM, Naito A, Schott M, Kim S, Ahlquist P.
Involvement of host cellular multivesicular body functions in hepatitis B virus

U
budding. Proceedings of the National Academy of Sciences of the United States of
America. 2007;104(24):10205-10.
AN
63. Bruss V. Envelopment of the hepatitis B virus nucleocapsid. Virus research.
2004;106(2):199-209.
64. Prange R. Host factors involved in hepatitis B virus maturation, assembly, and
M

egress. Medical microbiology and immunology. 2012;201(4):449-61.

65. Su TS, Lai CJ, Huang JL, Lin LH, Yauk YK, Chang CM, et al. Hepatitis B
virus transcript produced by RNA splicing. Journal of virology. 1989;63(9):4011-8.
D

66. Suzuki T, Masui N, Kajino K, Saito I, Miyamura T. Detection and mapping of

spliced RNA from a human hepatoma cell line transfected with the hepatitis B virus
genome. Proceedings of the National Academy of Sciences of the United States of
TE

America. 1989;86(21):8422-6.
67. Terre S, Petit MA, Brechot C. Defective hepatitis B virus particles are
generated by packaging and reverse transcription of spliced viral RNAs in vivo.
EP

Journal of virology. 1991;65(10):5539-43.

68. Gunther S, Sommer G, Iwanska A, Will H. Heterogeneity and common
features of defective hepatitis B virus genomes derived from spliced pregenomic
C

RNA. Virology. 1997;238(2):363-71.

69. Sommer G, van Bommel F, Will H. Genotype-specific synthesis and secretion
AC

of spliced hepatitis B virus genomes in hepatoma cells. Virology. 2000;271(2):371-

81.
70. Lee GH, Wasser S, Lim SG. Hepatitis B pregenomic RNA splicing--the
products, the regulatory mechanisms and its biological significance. Virus research.
2008;136(1-2):1-7.
71. Huang ZM, Yen TS. Role of the hepatitis B virus posttranscriptional
regulatory element in export of intronless transcripts. Mol Cell Biol.
1995;15(7):3864-9.
72. Rosmorduc O, Petit MA, Pol S, Capel F, Bortolotti F, Berthelot P, et al. In
vivo and in vitro expression of defective hepatitis B virus particles generated by
spliced hepatitis B virus RNA. Hepatology. 1995;22(1):10-9.

25
 ACCEPTED MANUSCRIPT
73. Soussan P, Tuveri R, Nalpas B, Garreau F, Zavala F, Masson A, et al. The
expression of hepatitis B spliced protein (HBSP) encoded by a spliced hepatitis B
virus RNA is associated with viral replication and liver fibrosis. Journal of
hepatology. 2003;38(3):343-8.
74. Marschenz S, Endres AS, Brinckmann A, Heise T, Kristiansen G, Nurnberg P,
et al. Functional analysis of complex hepatitis B virus variants associated with
development of liver cirrhosis. Gastroenterology. 2006;131(3):765-80.
75. Girones R, Miller RH. Mutation rate of the hepadnavirus genome. Virology.
1989;170(2):595-7.

PT
76. Okamoto H, Tsuda F, Sakugawa H, Sastrosoewignjo RI, Imai M, Miyakawa
Y, et al. Typing hepatitis B virus by homology in nucleotide sequence: comparison of
surface antigen subtypes. The Journal of general virology. 1988;69 ( Pt 10):2575-83.

RI
77. Norder H, Courouce AM, Magnius LO. Complete genomes, phylogenetic
relatedness, and structural proteins of six strains of the hepatitis B virus, four of which
represent two new genotypes. Virology. 1994;198(2):489-503.

SC
78. Davies J, Littlejohn M, Locarnini SA, Whiting S, Hajkowicz K, Cowie BC, et
al. Molecular epidemiology of hepatitis B in the Indigenous people of northern
Australia. Journal of gastroenterology and hepatology. 2013;28(7):1234-41.
79. Sugauchi F, Kumada H, Acharya SA, Shrestha SM, Gamutan MT, Khan M, et

U
al. Epidemiological and sequence differences between two subtypes (Ae and Aa) of
hepatitis B virus genotype A. The Journal of general virology. 2004;85(Pt 4):811-20.
AN
80. Locarnini S, Hatzakis A, Chen DS, Lok A. Strategies to control hepatitis B:
Public policy, epidemiology, vaccine and drugs. Journal of hepatology. 2015;62(1
Suppl):S76-86.
M

81. Suzuki Y, Kobayashi M, Ikeda K, Suzuki F, Arfase Y, Akuta N, et al.

Persistence of acute infection with hepatitis B virus genotype A and treatment in
Japan. Journal of medical virology. 2005;76(1):33-9.
D

82. Matsuura K, Tanaka Y, Hige S, Yamada G, Murawaki Y, Komatsu M, et al.

Distribution of hepatitis B virus genotypes among patients with chronic infection in
Japan shifting toward an increase of genotype A. Journal of clinical microbiology.
TE

2009;47(5):1476-83.
83. Zhang HW, Yin JH, Li YT, Li CZ, Ren H, Gu CY, et al. Risk factors for acute
hepatitis B and its progression to chronic hepatitis in Shanghai, China. Gut.
EP

2008;57(12):1713-20.
84. Chu CM, Liaw YF. Genotype C hepatitis B virus infection is associated with a
higher risk of reactivation of hepatitis B and progression to cirrhosis than genotype B:
C

a longitudinal study of hepatitis B e antigen-positive patients with normal

aminotransferase levels at baseline. Journal of hepatology. 2005;43(3):411-7.
AC

85. Kao JH, Chen PJ, Lai MY, Chen DS. Genotypes and clinical phenotypes of
hepatitis B virus in patients with chronic hepatitis B virus infection. Journal of clinical
microbiology. 2002;40(4):1207-9.
86. Livingston SE, Simonetti JP, Bulkow LR, Homan CE, Snowball MM, Cagle
HH, et al. Clearance of hepatitis B e antigen in patients with chronic hepatitis B and
genotypes A, B, C, D, and F. Gastroenterology. 2007;133(5):1452-7.
87. Chan HL, Hui AY, Wong ML, Tse AM, Hung LC, Wong VW, et al. Genotype
C hepatitis B virus infection is associated with an increased risk of hepatocellular
carcinoma. Gut. 2004;53(10):1494-8.
88. Chu CJ, Hussain M, Lok ASF. Hepatitis B virus genotype B is associated with
earlier HBeAg seroconversion compared with hepatitis B virus genotype C.
Gastroenterology. 2002;122(7):1756-62.

26
 ACCEPTED MANUSCRIPT
89. Sanchez-Tapias JM, Costa J, Mas A, Bruguera M, Rodes J. Influence of
hepatitis B virus genotype on the long-term outcome of chronic hepatitis B in western
patients. Gastroenterology. 2002;123(6):1848-56.
90. Kao JH, Wu NH, Chen PJ, Lai MY, Chen DS. Hepatitis B genotypes and the
response to interferon therapy. Journal of hepatology. 2000;33(6):998-1002.
91. Lim SG, Cheng Y, Guindon S, Seet BL, Lee LY, Hu P, et al. Viral quasi-
species evolution during hepatitis Be antigen seroconversion. Gastroenterology.
2007;133(3):951-8.
92. Fang ZL, Yang J, Ge X, Zhuang H, Gong J, Li R, et al. Core promoter

PT
mutations (A(1762)T and G(1764)A) and viral genotype in chronic hepatitis B and
hepatocellular carcinoma in Guangxi, China. Journal of medical virology.
2002;68(1):33-40.

RI
93. Kao JH, Chen PJ, Lai MY, Chen DS. Basal core promoter mutations of
hepatitis B virus increase the risk of hepatocellular carcinoma in hepatitis B carriers.
Gastroenterology. 2003;124(2):327-34.

SC
94. Qu LS, Zhu J, Liu TT, Shen XZ, Chen TY, Ni ZP, et al. Effect of combined
mutations in the enhancer II and basal core promoter of hepatitis B virus on
development of hepatocellular carcinoma in Qidong, China. Hepatology research : the
official journal of the Japan Society of Hepatology. 2014;44(12):1186-95.

U
95. Lyu H, Lee D, Chung YH, Kim JA, Lee JH, Jin YJ, et al. Synergistic effects
of A1896, T1653 and T1762/A1764 mutations in genotype c2 hepatitis B virus on
AN
development of hepatocellular carcinoma. Journal of viral hepatitis. 2013;20(3):219-
24.
96. Hunt CM, McGill JM, Allen MI, Condreay LD. Clinical relevance of hepatitis
M

B viral mutations. Hepatology. 2000;31(5):1037-44.

97. Brunetto MR, Giarin MM, Oliveri F, Chiaberge E, Baldi M, Alfarano A, et al.
Wild-type and e antigen-minus hepatitis B viruses and course of chronic hepatitis.
D

Proceedings of the National Academy of Sciences of the United States of America.

1991;88(10):4186-90.
98. Yuen MF, Tanaka Y, Ng IO, Mizokami M, Yuen JC, Wong DK, et al. Hepatic
TE

necroinflammation and fibrosis in patients with genotypes Ba and C, core-promoter

and precore mutations. Journal of viral hepatitis. 2005;12(5):513-8.
99. Yuen MF, Sablon E, Yuan HJ, Hui CK, Wong DK, Doutreloigne J, et al.
EP

Relationship between the development of precore and core promoter mutations and
hepatitis B e antigen seroconversion in patients with chronic hepatitis B virus. The
Journal of infectious diseases. 2002;186(9):1335-8.
C

100. Sonneveld MJ, Rijckborst V, Zeuzem S, Heathcote EJ, Simon K, Senturk H,

et al. Presence of precore and core promoter mutants limits the probability of response
AC

to peginterferon in hepatitis B e antigen-positive chronic hepatitis B. Hepatology.

2012;56(1):67-75.
101. Bayliss J, Yuen L, Rosenberg G, Wong D, Littlejohn M, Jackson K, et al.
Deep sequencing shows that HBV basal core promoter and precore variants reduce
the likelihood of HBsAg loss following tenofovir disoproxil fumarate therapy in
HBeAg-positive chronic hepatitis B. Gut. 2016.
102. Carman WF, Zanetti AR, Karayiannis P, Waters J, Manzillo G, Tanzi E, et al.
Vaccine-induced escape mutant of hepatitis B virus. Lancet. 1990;336(8711):325-9.
103. Thakur V, Kazim SN, Guptan RC, Hasnain SE, Bartholomeusz A, Malhotra
V, et al. Transmission of G145R mutant of HBV to an unrelated contact. Journal of
medical virology. 2005;76(1):40-6.

27
 ACCEPTED MANUSCRIPT
104. Chakravarty R, Neogi M, Roychowdhury S, Panda CK. Presence of hepatitis
B surface antigen mutant G145R DNA in the peripheral blood leukocytes of the
family members of an asymptomatic carrier and evidence of its horizontal
transmission. Virus research. 2002;90(1-2):133-41.
105. Carman WF, Korula J, Wallace L, MacPhee R, Mimms L, Decker R.
Fulminant reactivation of hepatitis B due to envelope protein mutant that escaped
detection by monoclonal HBsAg ELISA. Lancet. 1995;345(8962):1406-7.
106. Basuni AA, Butterworth L, Cooksley G, Locarnini S, Carman WF. Prevalence
of HBsAg mutants and impact of hepatitis B infant immunisation in four Pacific

PT
Island countries. Vaccine. 2004;22(21-22):2791-9.
107. Kamili S, Sozzi V, Thompson G, Campbell K, Walker CM, Locarnini S, et al.
Efficacy of hepatitis B vaccine against antiviral drug-resistant hepatitis B virus

RI
mutants in the chimpanzee model. Hepatology. 2009;49(5):1483-91.
108. Warner N, Locarnini S. The antiviral drug selected hepatitis B virus
rtA181T/sW172* mutant has a dominant negative secretion defect and alters the

SC
typical profile of viral rebound. Hepatology. 2008;48(1):88-98.
109. Sugauchi F, Ohno T, Orito E, Sakugawa H, Ichida T, Komatsu M, et al.
Influence of hepatitis B virus genotypes on the development of preS deletions and
advanced liver disease. Journal of medical virology. 2003;70(4):537-44.

U
110. Chen CH, Hung CH, Lee CM, Hu TH, Wang JH, Wang JC, et al. Pre-S
deletion and complex mutations of hepatitis B virus related to advanced liver disease
AN
in HBeAg-negative patients. Gastroenterology. 2007;133(5):1466-74.
111. Hsieh YH, Su IJ, Wang HC, Tsai JH, Huang YJ, Chang WW, et al. Hepatitis
B virus pre-S2 mutant surface antigen induces degradation of cyclin-dependent kinase
M

inhibitor p27Kip1 through c-Jun activation domain-binding protein 1. Mol Cancer

Res. 2007;5(10):1063-72.
112. Ito K, Tanaka Y, Kato M, Fujiwara K, Sugauchi F, Sakamoto T, et al.
D

Comparison of complete sequences of hepatitis B virus genotype C between inactive

carriers and hepatocellular carcinoma patients before and after seroconversion.
Journal of gastroenterology. 2007;42(10):837-44.
TE

113. Kajiya Y, Hamasaki K, Nakata K, Nakagawa Y, Miyazoe S, Takeda Y, et al.

Full-length sequence and functional analysis of hepatitis B virus genome in a virus
carrier: a case report suggesting the impact of pre-S and core promoter mutations on
EP

the progression of the disease. Journal of viral hepatitis. 2002;9(2):149-56.

114. Lai CL, Dienstag J, Schiff E, Leung NW, Atkins M, Hunt C, et al. Prevalence
and clinical correlates of YMDD variants during lamivudine therapy for patients with
C

chronic hepatitis B. Clinical infectious diseases : an official publication of the

Infectious Diseases Society of America. 2003;36(6):687-96.
AC

115. Marcellin P, Chang TT, Lim SG, Sievert W, Tong M, Arterburn S, et al.
Long-term efficacy and safety of adefovir dipivoxil for the treatment of hepatitis B e
antigen-positive chronic hepatitis B. Hepatology. 2008;48(3):750-8.
116. Chang TT, Lai CL, Kew Yoon S, Lee SS, Coelho HS, Carrilho FJ, et al.
Entecavir treatment for up to 5 years in patients with hepatitis B e antigen-positive
chronic hepatitis B. Hepatology. 2010;51(2):422-30.
117. Zoulim F, Locarnini S. Hepatitis B virus resistance to nucleos(t)ide analogues.
Gastroenterology. 2009;137(5):1593-608 e1-2.
118. Liu Y, Corsa AC, Buti M, Cathcart AL, Flaherty JF, Miller MD, et al. No
detectable resistance to tenofovir disoproxil fumarate in HBeAg+ and HBeAg-
patients with chronic hepatitis B after 8 years of treatment. Journal of viral hepatitis.
2017;24(1):68-74.

28
 ACCEPTED MANUSCRIPT
119. Lee JH, Han KH, Lee JM, Park JH, Kim HS. Impact of hepatitis B virus
(HBV) x gene mutations on hepatocellular carcinoma development in chronic HBV
infection. Clinical and vaccine immunology : CVI. 2011;18(6):914-21.
120. Sung WK, Zheng H, Li S, Chen R, Liu X, Li Y, et al. Genome-wide survey of
recurrent HBV integration in hepatocellular carcinoma. Nature genetics.
2012;44(7):765-9.

PT
RI
U SC
AN
M
D
TE
C EP
AC

29
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
Figure 1: The HBV life cycle. HBV enters the hepatocyte via the sodium taurocholate cotransporting
polypeptide (NTCP) receptor. The nucleocapsid is uncoated and migrates to the nucleus where it delivers the
genome (rc-DNA). Rc-DNA is repaired to form double-stranded cccDNA and a mini chromosome from which
the following HBV viral proteins are transcribed: core and precore antigen, surface antigens S, M and L, X
M

protein and polymerase. After processing by the endoplasmic reticulum (ER), HBeAg and HBsAg are secreted.
New virions are released and genomic RNA is recycled back into the nucleus for ongoing viral replication.
D
TE
C EP
AC