HBV - Virology

Introduction

In 1965, Blumberg and colleagues - in search of tools to identify and track genetic
differences in different human populations – found a novel antigen present in sera
of Australian Aborigines (Blumberg 1965). This antigen was preliminarily named
Australia antigen and was associated with a clinical course of hepatitis in the following
years (Blumberg 1967; Blumberg 1968) and immediately thereafter to one
specific type called serum hepatitis (Okochi 1968; Okochi 1970; Okochi 1993).
Electron microscopy studies revealed that patients testing positive for the Australia
antigen had two different types of particles in their serum that contained the Australia
antigen, namely small particles of spherical and rod-like shapes with a diameter
of around 22 nm, and the so-called Dane particles, of 42 nm (Dane 1970),
which are the intrinsic infectious virus particles containing the viral genome and are
named human hepatitis B virus (HBV) (Heermann 1984; Kaplan 1973; Robinson
1974; Robinson 1974; Robinson 1975b; Robinson 1975a; Robinson 1976b; Robinson
1976a). Meanwhile, it turns out that a number of HBV-like viruses exist, most
of them displaying a very narrow host range; altogether, these viruses form the viral
family Hepadnaviridae.

Taxonomic classification of the Hepadnaviridae

The family name Hepadnaviridae is based on the clinical picture of infection and
the target organ (liver, classical/ancient Greek: to hepar) and its nucleic acid, DNA.
The family of Hepadnaviridae contains two genera, the orthohepadnaviruses that
infect only mammals, and the avihepadnaviruses that infect birds.
Taxonomically, the Hepadnaviridae form their own group because of biological
characteristics not observed in any other viral family known to date. The Hepadnaviridae
contain one of the smallest pathogen genomes known, of just 3-3.3 kbp. The
reading frames on the genome are organized in a unique and highly condensed way
and overlap, which contributes to a unique replication strategy. This strategy includes
a reverse transcription step that is also observed in the replication of retroviruses,
but, in contrast to retroviruses, the nucleic acid packaged into hepadnaviral
infectious particles is DNA, not the RNA of retroviruses.
The sub-classification into the two genera is based on the differences in the host and
additionally on phylogenetic differences between mammalian and avian hepadnaviruses
(Figure 1). Until now, two major species have been assigned to the avihepadnaviruses
and were named after their individual hosts, namely the duck hepatitis B
virus (DHBV) and the heron hepatitis B virus (HHBV). Additionally, a number of
other avihepadnaviruses have been described that have not been specifically categorized
(Guo 2005). On the other hand, the orthohepadnavirus genus includes the four
best-known distinct species - HBV, WHV, GSHV and WMHV. The prototype species
is the human hepatitis B virus (HBV) that infects humans and can be used to
experimentally infect chimpanzees. WHV, the woodchuck hepatitis virus, is a wellstudied
orthohepadnavirus that occurs naturally in marmots and cannot be trans
ferred to other rodents like its relative GSHV, the ground squirrel hepatitis virus.
Interestingly, GSHV can also infect woodchucks, thus its host range is not as narrow
as the WHV host range. The last species on the list, the woolly monkey hepatitis
B virus (WMHV), despite having a non-human primate as its natural host, in
contrast to HBV, is not infectious for chimpanzees (Lanford 1998; Lanford 2003;
Seeger 1987; Seeger 1991). A further member of the genus, the arctic ground squirrel
hepatitis virus (AGSHV) is most closely related to GSHV, but has not been further
assigned, as studies on its host range have not been published yet (Testut
1996). Hepadnavirus isolates from chimpanzees, gorillas, orangutans, and gibbons
were initially believed to be distinct species but are currently considered to be HBV
subtypes rather than distinct species (Testut 1996; Verschoor 2001; Warren 1999;
Thornton 2001; Zuckerman 1975; Zuckerman 1978; Hu 2000; Starkman 2003). In
humans, HBV is divided into eight genotypes, A-H; however, it cannot be excluded
that other genotpyes will occur or evolve in the future. Genotypes A-H display
pairwise differences of between 8 and 17% (Fung 2004; Norder 1994; Norder 2003;
Arauz-Ruiz 2002; Arauz-Ruiz 2001; Arauz-Ruiz 1997b; Arauz-Ruiz 1997a).
Abbreviations. AGSHV=arctic ground squirrel hepatitis virus, ASHBV=ashy headed
sheldgoose HBV, CHBV=crane HBV, ChHBV=Chimpanzee HBV, GiHBV=Gibbon HBV,
GoHBV=Gorilla HBV, GSHV=ground squirrel hepatitis virus, CWHBV=chileo wigeon HBV,
HHBV=heron HBV, OSHBV=Orinoco sheldgoose HBV, OuHV=Orangutan hepadnavirus,
PTHBV=puna teal HBV, RGHBV=Ross’ goose HBV, SGHBV=snow goose HBV,
STHBV=storck HBV, WHV=woodchuck hepatitis virus, WMHBV=woolly monkey HBV

Structure of virus particles and organization of the viral genome

The Hepadnaviridae are enveloped DNA viruses with a circular partially doublestranded
DNA that in concert with the core protein forms the nucleocapsid. The
infectious virus, i.e., the Dane particle, displays a spherical shape with a diameter of
42-47 nm. The viral membrane that is acquired by the virus during budding or
while the viral particles are transported through secretory pathways via the endoplasmatic
reticulum and Golgi pathways forms the surface containing three viral
surface proteins. These proteins, according to their size, named HB small surface
antigen (HBsAg), middle (HBMAg), or large (HBLAg), are acquired during budding
into the endoplasmatic reticulum (ER).
The nucleocapsid, which forms the inner part of the Dane particle, is around 28 nm
in size and besides a single copy of viral genome contains the viral polymerase,
which is covalently bound to the viral genome, and in turn leads to problems in the
molecular diagnostics of HBV infections (see Chapter 8). As with nearly all enveloped
viruses there is also evidence that the HBV particle contains proteins assumed
to be of host origin (Albin 1980).
The average size of the viral genome is around 3.3 kbp, varying slightly from
genotype to genotype and from isolate to isolate. Figure 2 shows the open reading
frame organization of the HBV genome. All open reading frames are in an identical
orientation and overlap at least partially. Within the Dane particle the negative
strand of the viral genome is present in full-length, thus carrying the whole genome.
In contrast, the positive strand spans only ~ 2/3 of the genome in length, whilst its
3’-end is variable in size (Lutwick 1977; Summers 1975). The viral polymerase is
covalently bound to the negative strand by a phosphotyrosine bond. At the 5’-end of
the positive strand a short RNA oligomer originating from the pre-genomic (pg)
RNA residually remains bound covalently after the viral DNA synthesis. The negative
strand, in contrast to the positive strand, contains on both the 5’-end and the 3’-
end a small redundancy of 8-9 nucleotides in length, named the r-region. These
redundant structures are essential for viral replication (Seeger 1986; Will 1987;
Lien 1986a; Lien 1987).
The viral genome covers four open reading frames, all of them encoded by the
negative strand, with 6 start codons, four promoters, two transcription enhancing
elements, a poly-adenylation signal motif, and a number of signals for DNA replication
(Figure 2). The major RNA transcripts are polyadenylated, capped, 3.5 kb,
2.4 kb, and 2.1 kb in length and named pre-C/C, preS, and S mRNAs (Enders 1985;
Cattaneo 1984). Moreover, a 0.7 kb long mRNA termed X mRNA occurs occasionally.
The 3’-end of all HBV transcripts is common for all of them and created by
the polyadenylation signal in the core (C) gene.
The viral genome encodes for the core protein, the pre-core protein also known as
the e-antigen, the polymerase, the three surface proteins, and the X protein. Whilst
the core protein – that is also recognized by the immune system – is essential for the
formation of nulceocapsids, the e-antigen that also contains the full core gene, is
post-translationally processed and as a non-essential gene is important in the viralhost
immunity interaction. E-antigen is also a marker for active viral replication and
plays an important role in molecular diagnostics (Chen 2004).
Figure 2. Genome organization and transcripts of the human hepatitis B virus.
The viral polymerase is the single enzyme encoded by the HBV genome and is an
RNA-dependent DNA polymerase with RNaseH activity. The HBV polymerase
consists of three functional domains and a so-called spacer region; the terminal
protein (TP) is located at its N-terminal domain, acting as a primer in negativestrand
DNA synthesis. The C-terminal region is separated by the spacer and functions
as the RT-polymerase and the RNaseH.
The HBV replication cycle 59
The three surface proteins, L, M, and S, share the C-terminal s-domain and are
coded on one open reading frame that encodes three start codons (one for L: preS1,
one for M: preS2, and one for S: preS3) and overlaps with the polymerase open
reading frame (Seeger 2007). So far, the role of the X protein is not fully understood,
although it has been associated with the nucleus and the cytoskeleton (Doria
1995; Henkler 2001; Lara-Pezzi 2001). However, HBX (hepatitis B X protein) is
required for efficient infection in vivo (Zhang 2001; Zoulim 1994).

The HBV replication cycle

Despite 40 years of HBV research, no widely available cell lines permissive for
HBV or any other member of the Hepadnaviridae family has been described. Either
studies on the replication cycle of Hepadnaviridae, i.e., attachment, entry, genome
replication, transcription and expression of viral genes, assembly, and budding cannot
be fully executed, or studies are limited to small series of experiments with primary
permissive hepatocytes (Tuttleman 1986; Aldrich 1989; Ochiya 1989; Gripon
1993; Gripon 1988). Unfortunately, primary hepatocytes remain permissive for
only a short time after being harvested from the intact liver.
Yet it is assumed that viral entry and the host range of hepadnavirus is dependent
on the N-terminus of the large surface antigen (Ishikawa 1995; Chouteau 2001;
Lambert 1990; Gripon 2005; Urban 2002). So far, the intrinsic HBV receptor has
not been discovered, but from studies on DHBV in primary duck hepatocytes it is
assumed that around 104 receptor molecules per cell mediate the rapid binding,
followed by a slow uptake of the virus to the cell which can take up to 16 hours
(Pugh 1989; Klingmuller 1993; Pugh 1995; Rigg 1992; Hagelstein 1997; Kock
1996). Following entry into the hepatocyte and uncoating, which may proceed in
parallel, the nucleocapsid is transported into the cell’s nucleus, where the viral nucleic
acid is released. Release of the viral DNA and disintegration of the nucleocapsid
is assumed to take place at the nuclear core complex (Kann 1997; Rabe 2003).
In the infected hepatocyte the viral DNA is immediately transformed into the covalently
closed circular (ccc) DNA by cellular enzymes. The cccDNA in turn is the
template for transcription of viral genes, acts chemically and structurally as an episomal/
extrachromosomal DNA, and has a plasmid-like structure (Bock 1994; Bock
2001; Newbold 1995). Congruent with the fact that HBV infects hepatocytes,
nearly all elements regulating viral transcription have binding sites for liver-specific
transcription factors (Schaller & Fischer 1991; Lopez-Cabrera 1991; Lopez-Cabrera
1990; Guo 1993; Courtois 1987; Raney 1995). Nevertheless, although a number of
factors and interactions regulating viral transcription are known, the exact mechanisms
of HBV transcription remains unclear. However, it is known that viral transcription
occurs in the nucleus, and both messenger and pregenomic RNAs are
transported into the cytoplasm where they are respectively translated or used as the
template for progeny genome production.
In the cytoplasm, the core protein which itself can be phosphorylated by several
kinases, forms the basis for the nucleocapsid. It plays an active role in binding and
packaging of the pregenomic RNA, recruitment of the viral polymerase, and thus
enables the RT-polymerase/RNA complex to initiate reverse transcription within
the newly forming nucleocapsids (Liao 1995; Lan 1999; Gerlich 1982; Kann 1993;
Kau 1998; Daub 2002; Watts 2002).
The three surface proteins of HBV have two major properties. First, as transmembrane
proteins they are anchored in the viral envelope and thus are located on the
surface of the virus, being responsible for binding to the as yet unknown viral receptor.
Second, the three surface proteins are secreted as subviral particles that do
not contain a functional nucleocapsid. The proteins differ in their N-terminal sequences
that are longer than in the cases of the L and M protein. All proteins have
in common the S domain; the M additionally has the pre-S2 domain; the L has both
the pre-S2 and the pre-S1 domain (Figure 2). The surface proteins of mammalian
Hepdnaviridae have been shown to be N- and O-glycosylated (Schildgen 2004; Lu
2003; Block 1998; Block 1994; Schmitt 2004; Schmitt 1999). These glycosylations
have been shown to be responsible for proper secretion of progeny viral particles
and in turn may represent novel targets for therapies with glycosylation inhibitors
(Schildgen 2004; Lu 2003; Block 1998; Block 1994; Schmitt 2004; Schmitt 1999).
Moreover, the surface proteins have been demonstrated to be activators of transcription
by acting individually (Kekule 1990; Caselmann 1990).
The viral polymerase, the only enzyme encoded by the hepadnaviral genome, consists
of three functional domains – the terminal protein, the reverse transcriptase,
and the RNaseH domain – and a spacer domain that separates the terminal protein
from the polymerase domains. The terminal protein also serves as a primer for reverse
transcription (Wang 1992; Weber 1994; Lanford 1997). Before or during
formation of the cccDNA the terminal protein and one of the redundant terminal
repeats present on the relaxed circular viral genomic DNA that is released from the
nucleocapsid are removed and the cccDNA forms via a not fully understood
mechanism, most probably dependent on cellular ligases and maybe other enzymes.
So far it is assumed that cellular DNA repair mechanisms become active and convey
the relaxed circular form into the cccDNA (Seeger 2007).
As mentioned previously, the cccDNA also is the template for the pre-genomic
RNA (pgRNA). This RNA is both the template for core and polymerase protein
translation and is the matrix for the progeny genomes as well. The pgRNA bears a
secondary structure - named å-structure - that is present at both the 5’- and the 3’-
ends. The å-hairpin loops at the 5’-end are first recognized by the viral polymerase
and act as the initial packaging signal (Bartenschlager 1992; Hirsch 1990; Huang
1991). The synthesis of the DNA negative strand, i.e., the intrinsic reverse transcription,
is then initiated by the formation of a covalent bond between the tyrosine
Y65 residue of the terminal protein domain and a desoxy-guanosinemonophosphate
(dGMP) (Wang 1992; Weber 1994; Lanford 1999; Zoulim 1994).
The next few nucleotides following this initial dGMP complement a small part of
the å-structure. The small terminal protein bound primer is subsequently translocated
to the 3’-end by an unknown mechanism but remains covalently bound the
whole time. This process is possibly a prerequisite for the correct folding of the
progeny genome within the newly forming nucleocapsid. Finally, the negative
strand is fully synthesized by the reverse transcription reaction while the RNA is
degraded by the RNaseH activity of the enzyme. The following positive strand
synthesis is initiated by an 18mer capped RNA oligo that remains from the 5’-end
of the pgRNA (Lien 1986b; Loeb 1991). Nevertheless, it is assumed that, although
not actively replicating and with conflicting data on its stability, there is evidence
that cccDNA may be stable in infected hepatocytes, thus contributing to chronic
HBV infection. This points to another possible target, long-term therapies that support
the elimination of cccDNA positive cells.
The final replication step, the assembly and release of HBV Dane particles, is not
fully understood; one study on usage of glycosylation inhibitors at non-toxic doses
suppressing the viremia in WHV infected woodchucks is indirect evidence that assembly
and release occur via secretory pathways (Block 1998).

Pathogenesis of hepadnavirus infections

The transmission of HBV and other members of the Hepadnaviridae family occur
vertically and horizontally via exchange of body fluids. In serum, a maximum of
1010 to 1012 genome copies per ml serum or body fluid can be found. In chronic
infections, the viremia is subject to natural fluctuations of +/- one log10 (Schildgen
2006).
The rate for chronicity, depending on the study, is >90% in neonatal infections and
approximately 10-15% in adult infections. The risk for transfusion-acquired and
nosocomial infections in the past two decades has decreased due to optimized molecular
diagnostics and more strict hygiene and legal regulations; however, there is
still a remarkable number of such transmissions due to incautious behavior of
healthcare personnel.
Once having entered the host, HBV reaches its major target cell, the hepatocyte, the
main site for replication and persistence, as virtually all hepadnaviruses display a
pronounced and distinct liver tropism. Furthermore, other cell types have been
shown to serve as non-hepatic reservoirs for mammalian hepadnaviruses. Within
the infected liver in immunocompetent hosts there is a continued damage of infected
hepatocytes by cytotoxic T lymphocytes (CTLs) that leads to uninterrupted
expression of collagen fibres, and in the worst and untreated cases to liver cirrhosis
(Pinzani 1995; Mathew 1996; Papatheodoridis 2005; Yoshida 2004; Rockey 2005;
Liaw 2004; Rizzetto 2005; Maynard 2005).
In this context it is worthwhile noting that there is no evidence that HBV is cytotoxic
for the infected hepatocyte. In contrast to other viruses that can infect the liver
like herpes simplex virus (HSV), HBV is unable to induce cytopathic effects under
normal infection conditions (Jilbert 1992; Kajino 1994; Wieland 2004; Thimme
2003). Liver damage (fibrosis, cirrhosis, and probably hepatocellular carcinoma) is
believed to be induced by the ongoing immune reaction and a consistent inflammation
of the liver.
Consequently, and confirmed by experimental data (Ando 1994; Guidotti 1994a;
Guidotti 1994b; Guidotti 1996; Guidotti 1999a; Guidotti 1999b; Guidotti 2000;
Kakimi 2001; Tsui 1995), it is generally assumed that massive CTL and NK-T cell
action resulting in killing of infected hepatocytes is essential for elimination of the
infection. It is further assumed that in those cases in which a chronicity of infection
evolves, the initial cellular immune response is too weak and thus not sufficient to
control the infection (Ganem 2004). Until now it has remained unclear which
mechanisms are responsible for the passage from the acute phase to the chronic
phase of the infection, thus this part of the viral life cycle remains a matter of
speculation. As a matter of fact, it has been shown that a sufficient Th1 response
involving CD8 positive CTLs, natural killer T cells (NK-T), cytokines (TNF-alpha,
other interferon gamma like IL-12, IL-15, etc.) is involved in suppression of transient
infections (Seeger 2007).
Despite the fact that only antibodies against the S protein are neutralizing and are
the major marker for immunity, it has been hypothesized that transient infection is
kept in check by gamma interferon and other cytokines released by immune cells,
leading in turn to a shutdown of viral replication (Schultz 1999; Pasquetto 2002;
Schultz 1999). However, this does not explain why antibodies against HBsAg are
present only in those patients who clear the virus; it is assumed to be a continuous
control of the infection. cccDNA can be found in those patients for decades
(Maynard 2005; Werle-Lapostolle 2004), whereas the above described mechanisms
fail if the infection passes to the chronic stage.

Animal models for HBV infections

As mentioned above it is crucial to make use of suitable model systems to study the
biology and clinical features of viral infection. Unfortunately, due to its narrow host
range this option is limited when studying HBV, because HBV refuses to replicate
other than in primary hepatocytes. Consequently it has been attempted by researchers
all over the world to establish animal models and cell culture systems that at
least partially reproduce some stages of HBV infection and can be used, e.g., for the
preclinical testing of novel antiviral drugs.

Chimpanzees

Both from epidemiological studies in captive animals and on the natural reservoirs
of hepadnaviridae as well as from experimental infection experiments it is known
that chimps and other higher human primates can be infected with HBV. Chimpanzees
have been used for preclinical testing of preventive and therapeutic vaccines
(Kim 2008; Komiya 2008; Murray 2005; Sallberg 1998; Pride 1998; Ogata 1993;
Wahl 1989; Lubeck 1989; Sureau 1988; Acs 1987; Will 1983). Fortunately, for
ethical, economic and scientific reasons, experiments with chimpanzees have been
nearly eliminated.

Woodchucks and squirrels

The woodchuck turned out to be a model for HBV infections by a lucky chance at
the end of the 1970s. In the Philadelphia Zoo, where the Penrose Research Laboratory
was located, it was observed that woodchucks captured in the mid-Atlantic
states of the US and housed in the Philadelphia Zoo frequently suffered from hepatocellular
carcinoma (Summers 1978). In contrast, in the woodchuck population
trapped in New York and its countryside no hepatomas were observed. The hepatocellular
carcinoma was associated with an HBV-like virus, termed woodchuck
hepatitis virus (WHV). WHV surprisingly cannot infect European marmots. The
woodchuck and its woodchuck hepatitis virus is an accepted model for HBV for
preclinical testing for novel antiviral drugs.
In spite of a number of advantages the woodchuck model is not very widespread.
Despite some successful attempts to breed woodchucks under laboratory conditions
as done at Cornell University, where a woodchuck colony is housed, most laboratories
have failed to breed or only by chance have managed to breed a limited number
of woodchucks. Consequently, most labs have to rely on wild captured animals with
or without chronic infections, which entails complications. Wild captured animals
bear the risk of being infected with other pathogens such as parasites or the rabies
virus, may carry ectoparasites or other unknown comorbidities. Furthermore, research
with wild captured animals requires special permission, at least in Europe,
independent of the fact that these animals may be captured as agrarian varmints,
thus resulting in an overwhelming bureaucracy with customs and local legal
authorities. Moreover, hibernation may influence experiments, especially if after
being captured woodchucks remain in this mode. Finally, as wild captured woodchucks
weigh up to 7 kg and are not willing to assist the researcher in the planned
experiments, they have to be anesthetized before any manipulation is performed.
However, the number of secondary reagents needed for woodchuck research,
though not commercially available, is increasing and will be a useful tool in future
research.
The ground squirrel hepatitis virus was detected shortly after WHV (Marion 1980)
and like WHV but unlike DHBV can induce hepatocellular carcinoma. In their
natural host GSHV seems to be less severe than WHV in woodchucks (Marion
1983; Marion 1983; Marion 1986; Cullen 1996).

Ducks

Within the genus of avihepadnaviruses, the duck hepatitis virus that infects the domestic
duck was the first species described (Mason 1980). Surprisingly, in birds the
hepadnavirus infection is totally apathogenic, likely because DHBV spread occurs
vertically in most cases. The viral replication of DHBV in its host takes place in the
yolk sac, liver, spleen, kidney, and pancreas. Although some aspect of orthohepadnavirus
infections can be studied with this model, the model has limitations. In
contrast to what is observed in mammals, avihepadnaviruses have not yet been associated
with liver damage as a consequence of infections, i.e., fibrosis, cirrhosis
and subsequent carcinoma do not develop during chronic infection.
Moreover, up to 50% of ducks develop a liver disease unrelated to DHBV that may
overlap DHBV-induced effects. Furthermore, if not transmitted vertically, DHBV
infection is cleared within a few days post-infection in contrast to mammalian
hepadnaviruses. Finally, despite the fact that the duck model was widely used in
preclinical trials (e.g., Chen 2007; Foster 2005; Le Guerhier 2003; Deres 2003;
Kumar 2002; Delmas 2002; Kumar 2001; Kumar 2001; Kumar 2001; Seigneres
2001; Peek 2001; Chu 1998; Xin 1998; Seifer 1998; Offensperger 1996; Hafkemeyer
1996; Lofgren 1996; Heijtink 1993) it has to be kept in mind that birds do
have a biology that differs from mammalian biology in many aspects, a problem
that has been lost sight of in the past.

Mouse models

Although some important aspects of HBV infections have been investigated in
transgenic mice and have led to results convincing the majority of HBV researchers,
these results have to be handled with care. In transgenic mice expression of
viral antigens is possible but does not necessarily reflect the situation of a natural
infection, thus some of the observed aspects may need to be considered artificial.
However, approaches using adenovirus vectors carrying the HBV genome may remain
a beneficial tool as at least some aspects of the infection can be studied in a
well-characterized in vivo model (Seeger 2007).
Another approach makes use of mouse chimera that consist of immunosuppressed
mice transplanted with human hepatocytes. These mice have been shown to be susceptible
to hepatitis B and C viruses (Dandri 2001a; Dandri 2001b; Mercer 2001).
The major advantage of this model is that primary human hepatocytes remain susceptible
to HBV for a long time, but unfortunately, these models require extremely
well-controlled breeding conditions that limit their broad use.

Tupaia

Tupaias, or tree shrews, belong to the zoological order of Scadentia with the two
families Tupalidae and Ptilocercidae. So far, 20 species in 5 genera have been described.
One species, Tupaia belangeri chinesis, has been found to be susceptible to
HBV (Su 1999; Yan 1996b; Yan 1996a). The tupaia is a relatively new model, but
as it is directly permissive for HBV it may be the model of choice in the future.

Cell culture models for in vitro phenotyping

Mutations within the polymerase gene can be detected by various methods such as
direct sequencing, line probe assay or clonal analysis. While the sequencing of PCR
products directly from patient serum or from cloned vectors gives information
about amino acid exchanges within the major population of a patient or a clone, a
line probe assay can simultaneously detect several co-existing HBV populations,
although only mutations which were included in the test probe can be found. However,
the quantification of minor populations needs to be refined, and their clinical
impact determined. As yet, no cell line that is fully permissive for HBV has been
identified and so a simple drug phenotyping system has not yet been established.
Consequently, it remains difficult to perform phenotypic tests for each individual
clinical resistance. For these reasons, in daily practice genotypic resistance testing
is the method of choice. Besides classical sequence analysis methods (commercial
or in-house), line probe assays rapidly deliver information on mutations in the viral
genome that are known to be associated with resistance. Cell culture assays for the
study of HBV drug resistance are only used for confirmation of newly observed
mutations that may play a role in antiviral resistance. These methods in general include
site-directed mutagenesis of replication-competent HBV genomes, exchange
of HBV genome fragments, PCR amplification of complete HBV genomes or
cloning of amplified HBV genomes, followed by subsequent transfection of these
genomes. HBV replication capacity and drug susceptibility are usually measured by
quantification of the different species of viral nucleic acids that form in the cell
culture system. In order to minimize variations in transient transfection assays, and
to allow a more reproducible measure of drug susceptibility, mutant HBV genomes
were integrated into permanent cell lines (Yang 2005) or into baculovirus for transfer
into mammalian cells (Delaney 2001). The latter methods are more laborious,
but take whole genome variability into account and allow cross-resistance testing
against various drugs (Delaney 2001; Zoulim 2006). Although transfection or
transduction of mutant HBV genomes allows their replication capacity and drug
susceptibility to be studied, viral fitness can be assessed only incompletely as the
early steps of infection – viral uptake and entry into the hepatocyte – cannot be investigated
in these systems. Furthermore, it is most important to note that there is
no standardization of the methods used worldwide, thus it is very difficult to compare
the level of resistance caused by the individually tested mutations in a quantitative
manner.