Hepatitis b virus gene mutations associated with HBeAg seroconversion 2

It was proven that the hepatitis B viral DNA encoded the surface protein, core protein of the Dane particle, a putative DNA polymerase and a protein of unknown function, designated the X

Chapter 1

Introduction

1.1 History

Although viral hepatitis is a disease of antiquity and epidemic jaundice is mentioned in the Talmud (fifth century B.C.), the infectious nature of the disease was not recognized until the end of the nineteenth century

The first description of ‘hepatitis B’ dates back to 1885 when Lurman, a public health officer in Bremen, Germany, gave a detailed report of an outbreak of jaundice that developed among workers of a local company vaccinated against smallpox with glycerinized human serum (1)

The features that distinguished what was earlier referred to as infectious hepatitis (IH) from serum hepatitis (SH) became more apparent as a result of numerous experiments with human volunteers conducted in the 1940s (2-4) These studies defined two immunologically distinct diseases that were characterized by different incubation periods and differed in modes of transmission For many years, the two types of hepatitis were described by different names In 1947, MacCallum proposed the name hepatitis A for the enterically transmitted form with a shorter period of incubation, and hepatitis B for the form transmitted through blood products and displaying a longer incubation period (5)

The hepatitis B virus was the first human hepatitis virus from which the proteins and genome could be identified and characterized No specific serological markers were identified until 1965 when Blumberg and colleagues described the Australian Antigen in leukemia sera that is now known as hepatitis B surface antigen [HBsAg] (6) An immunodiffusion precipitin line between the HBsAg present in the serum of an Australian Aborigine and the antibody to HBsAg in the sera of a United States

hemophiliac provided the first clue Subsequent studies revealed that this ‘Australian Antigen’ was specific to the sera of acute and chronic hepatitis B patients (6;7) Dane and colleagues discovered virus-like particles that carried this antigen on their surface in hepatitis B patients using immune electron microscopy in 1970 These particles were consequently considered to be ‘the’ hepatitis B virus The “Dane particles”, 42 nm in diameter, could be distinguished from excess surface antigen tubules and spherules, 22

nm in diameter (8) Soon after, a 28-nm inner body inside the particle (the nucleocapsid

or core) was isolated from the full 42-nm virion following detergent treatment that removed the envelope This finding represented the identification of an additional antigen-antibody system specific for Hepatitis B [HBcAg and anti-HBc] (9) Magnius and Espmark subsequently found a new ‘e’ antigen in HBsAg positive sera among chronic hepatitis B patients and only antibody to ‘e’ was found in many ‘e’ antigen negative carriers (10)

During the early seventies, there were many advances in HBV research In 1971,

Krugman et al found that a specific hepatitis B immune serum globulin preparation could prevent hepatitis B among children (11) In the same year, Krugman et al

accomplished the active vaccination for hepatitis B with a crude vaccine, a heat inactivated infectious serum In 1973, hepatitis B viral DNA polymerase activity was identified in human sera rich in Dane particles (12) As techniques for cloning and amplification of DNA became available by the end of the seventies and the early eighties, the virus genome was cloned and sequenced It was proven that the hepatitis B viral DNA encoded the surface protein, core protein of the Dane particle, a putative DNA polymerase and a protein of unknown function, designated the X protein In 1982,

Summers and Mason opened up a new phase in HBV research by characterizing the mechanism of HBV replication (13)

All these advances led to an explosive growth in information about the hepatitis B virus, development of serological and molecular diagnostic tests for hepatitis B, an understanding of the pathogenesis and natural history of HBV infection and approval of antiviral therapy for the treatment of chronic hepatitis B

1.2 The hepatitis B virus

The hepatitis B virus (HBV) has become one the most studied viruses in the world due to its high prevalence of infection and broad clinical consequences, which range from acute to chronic hepatitis, to liver cirrhosis and hepatocellular carcinoma

Human hepatitis B virus belongs to the hepadnavirus family, which is made up of the genus orthohepadnavirus with members found in mammals: HBV, woodchuck hepatitis virus (WHV), ground squirrel hepatitis virus (GSHV); and the genus avihepadnavirus which consists of duck hepatitis B virus (DHBV) and grey heron hepatitis B virus (HHBV) The name hepadnavirus reflects the hepatotropism of these DNA viruses HBV has a restricted host range and can only infect human and chimpanzee Recently, however, the HBV was found to be able to infect a small animal, tupaia (Tupaia belangeri, tree shrew) (14) Similarly, non-human hepadnaviruses can only infect the species that are very closely related to the respective host in evolution

1.2.1 The life cycle of HBV

The major steps in HBV DNA replication have been revealed over the past 15 years Like other viruses, the life cycle of HBV can be divided into several steps:

attachment of the virus to the host cells, entry into the cells, release of the viral genome,

Figure 1 Life cycle of HBV The virus, shown above, with its typical

relaxed circular DNA genome, enters the hepatocyte and the DNA is transported to the nucleus and processed to covalently closed circular DNA (cccDNA) that is the template for transcription of viral RNA As viral RNA enters the cytoplasm, one of the largest, pregenome RNA, serves as mRNA

for the viral reverse transcriptase (RT) and core protein synthesis RT binds in

cis to its own mRNA and this complex is packaged into immature viral nucleocapsids Following completion of minus strand DNA synthesis, and at least partial completion of plus strand DNA synthesis, the nucleocapsids bind

to viral envelope proteins, bud into the endoplasmic reticulum (ER) and, ultimately, exit the cell At low concentrations of viral envelope proteins, nucleocapsids are transported to the nucleus to amplify cccDNA copy number [Koshy R., 1998 (15)]

expression of viral gene products, replication of the viral genome, assembly of virions and finally release of the virus into the host circulation from the liver cells (Figure 1)

1.2.1.1 Attachment and entry into the host cells

The initial phase of HBV infection involves the attachment of mature virions onto the host cell membrane As there are no available cell lines that support HBV replication, the initial steps of HBV entry are still poorly understood Since only primary duck hepatocytes explanted freshly from the liver can support infection by DHBV, binding studies of duck hepatocyte membranes with DHBV demonstrated a specific interaction involving the pre-S domain of the surface protein recently Carboxypeptidase D has been identified to interact specifically with the DHBV virions and to mediate uptake into avian cells (16) Some studies suggest that the pre-S1 domain, involved in binding to cells and

S protein, may also be important in interacting with cells by binding the human liver protein-Annexin V (17)

Entry of the virus results from fusion of the viral and host membranes as the nucleocapsid is released into the cytoplasm The fusion of the viral envelope with the host cell membrane through a fusion-like peptide that presents on the pre-S2 domain of the middle size hepatitis B surface antigen seems to mediate this process (18) This usually hidden pre-S2 domain may become exposed after binding of the virion to the cell receptors However, receptors for HBV have not yet been defined More recently, an 80 kDa protein on the hepatocyte surface found to bind to human HBV was suggested to possibly function as a primary viral attachment site (19) The HBV penetrates the cell membrane through virus-cell fusion and then the viral envelope is removed After

uncoating, it is believed that the nucleocapsid is transported to the nuclear membrane of the cell although details of these processes are still unknown As the size of the core particles are so big, release of the genome must occur before import of the viral genome into the nucleus

1.2.1.2 HBV genome repair and transcription

All the hepadnaviruses replicate via reverse transcription The plus strand of HBV DNA is always incomplete within virus particles About 90% of the viral DNA is found

in a relaxed circular conformation with a short cohesive overlap between the 5’ ends of the two DNA strands, while the remainder of the viral genome has a linear conformation Both conformations are infectious even though only the former consistently gives rise to infectious progeny viruses (20;21) Replication of HBV requires conversion of the virion relaxed circular DNA into a double-stranded covalently closed circular DNA (cccDNA) Once the viral DNA is transported into the nucleus, its conversion to cccDNA is processed, including repair of the single stranded regions of plus strand DNA by viral or cellular polymerase, removal of the covalently attached protein from the minus strand and the oligoribonucleotides from the 5’ of plus strand, ligation of the nicks and super coiling of the HBV cccDNA Unlike the retroviruses, integration of HBV DNA into the host cell DNA is not required for HBV replication In fact, integration of HBV could disrupt at least one gene and also prevent transcription of functional super genomic size RNA due to linearization of the circular HBV DNA Once the cccDNA is formed, the host cellular RNA polymerase II, viral enhancers and promoters activate to produce several sets of HBV transcripts that are approximately 3.5, 2.4, 2.1 and 0.7 kilo bases

(kb) in length (Figure 2) All of these transcripts co-terminate at an identical polyadenylation site The smaller subgenomic 2.4, 2.1 and 0.7 kb transcripts serve as mRNAs for the translation of three surface proteins and x protein The 3.5 kb supergenomic transcripts are longer than the HBV genome and serve to produce e, core, and polymerase proteins and as pregenomic RNA (pgRNA) The pgRNA lacking the ATG start codon for e protein is chosen specifically by the HBV polymerase protein for packaging into the nucleocapsid and serves as the template for reverse transcription (22)

Figure 2 Transcripts and promoters The numerical designations on the

HBV genome [0-3,221 base pairs (bp)] are based on the HBV subtype adw2

S, C, X and P represent the genes encoding hepatitis B surface antigen, core/e antigen, x protein and polymerase PreS1, preS2, Xp and Cp represent the promoter elements for the corresponding genes The enhancer elements are designated as Enhancer (Enh) I and II An, the single polyadenylation site, is utilized by all the HBV RNAs The HBV transcripts are shown outside of the genome Each viral transcript is labeled with its respective length in kilobases and a poly A tail at the 3’ end This Figure is modified from [Koshy R., 1998 (15)]

Large hepatitis B surface protein [LHBs] synthesis occurs in the cytosol because

of the presence of an ER translocation signal peptide The 2.4 kb pre-S1 mRNA is used for LHBs translation but not for middle and small hepatitis B surface protein [MHBs and SHBs] (23;24) The 2.1 kb pre-S2 mRNA codes for MHBs and SHBs The MHBs is translated from the first start codon which has no optimal flanking bases, whereas the expression of SHBs starts at the second start codon with optimal initiation of protein synthesis This probably explains why MHBs is present at only less than 20% of the level

of the SHBs in the sera of the infected individuals (25;26) These surface proteins are expressed in excess and are able to assemble into 22 nm spheres and filaments that are secreted through the ER and Golgi apparatus

The smallest 0.7 kb mRNA is translated to HBx protein Sequence predictions suggest that the HBx is a cytosolic protein (27)

1.2.1.4 Viral reverse transcription

Once sufficient quantities of HBc protein are synthesized and at least one polymerase protein has been translated, these proteins assemble to form core particles, which contain the pregenomic RNA, hsp90 and protein kinase C It is believed that the encapsidation starts only when the viral polymerase binds to the stem-loop sequence (ε)

at the 5’ end of pgRNA and the C-terminus of the polymerase interacts with the hepatitis

B core protein The arginine-rich region of core protein and, consequently, the binding ability is essential for pregenomic RNA encapsidation (28)

RNA-HBV reverse transcription is thought to proceed only within the nucleocapsid and is initiated when encapsidation of the pgRNA occurs by complex interactions among the core protein, polymerase and host cellular factors (29) The pgRNA transcript has a terminally redundant sequence comprised of about 200 nucleotides including direct repeat (DR) 1 and the stem-loop at the 5’ end A second copy of DR1

is located near the 3’ end of the pgRNA transcript When the polymerase binds to the 5’ stem-loop of pgRNA, it reverse transcribes the bulge sequence of the stem-loop region for four bases by using the hydroxyl group of tyrosine 96 in its own amino terminal domain as the primer These few reverse transcribed nucleotides are complementary to those in the 3’ DR1 region The polymerase, together with its covalently bound, newly transcribed nucleotides, dissociates from the template and reanneals to the 3’ DR1 of pgRNA to continue reverse transcription (Figure 3) As the minus DNA elongates, the newly copied pgRNA template is degraded by the RNase

H activity of the polymerase protein, which is actually covalently attached to the growing minus DNA strand About 15-18 capped ribonucleotides at the 5’ end of the

pgRNA remain undegraded when minus DNA strand synthesis is completed The oligoribonucleotide cap, together with the polymerase, is translocated to the 5’ DR2 region on the minus DNA strand After annealing the oligoribonucleotides to the 5’ DR2 of the minus DNA, a 3’ hydroxyl group becomes available for priming of plus DNA strand synthesis (Figure 3) Typically, the plus DNA strand is not completed until re-entry of the HBV virion into a host cell, resulting in the single stranded gap normally seen in the packaged HBV DNA

Figure 3 Replication of the HBV gnome The virion DNA is brought to the

cell nucleus and converted to cccDNA which is then transcribed to various RNAs, one of which servers as template for the polymerase and core protein These two proteins assemble together with pgRNA to the replication complex The reverse transcription starts at the primase ( ) During elongation of the minus strand, the reverse transcriptase ( ) probably remains linked to the primase The RNase H cleaves the RNA of DNA/RNA hybrids, leaving a complete, single-stranded DNA minus strand The remaining 18 bases from the 5’ terminal part of the pgRNA are able to shift from DR1 to DR2, where they serve as primer for the DNA plus strand after a template switch of the reverse transcriptase Multiplication of the HBV genome occurs via transcription of the pgRNA [Zuckerman A J., 1998 (30)]

1.2.1.5 Virion formation

A number of core particles containing mature HBV DNA may migrate to the nucleus and release the HBV DNA which is then converted to cccDNA Other core particles are enveloped by the HBV surface proteins and secreted as complete viruses Core proteins assemble in the cytosol and the HBc proteins that contain mature HBV genomes seem to exhibit an affinity for intracellular membranes that contain inserted large surface protein molecules

Concurrent with DNA synthesis, the hepatitis B surface proteins aggregate in regions of the Golgi complex in a process that excludes host membrane proteins from the complex Regions high in SHBs protein, with minimal MHBs, are able to bud into the lumen of the ER to produce the secreted 22 nm subviral particles At the same time, the HBsAg undergoes glycosylation in the ER and Golgi SHBs and MHBs are produced much more than LHBs The completed nucleocapsid associates with the area of Golgi high in HBV surface proteins The pre-S1 and S domains of the LHBs are thought to interact with the core particles at the cytoplasmic face of Golgi, pulling the nucleocapsid into the forming vesicle, resulting in the mature 42 nm enveloped particle which is secreted by exocytosis from the cell (31)

1.2.1.6 Covalently closed circular DNA pool

The pool of HBV cccDNA in the nucleus has to be restored to ensure viral persistence as the half life of HBV cccDNA is only about 2 to 3 days (32) Two methods may be possible for restoration of the pool: new infection of the hepatocyte with HBV, or

a re-entry of viral DNA into the nucleus after release from newly formed mature viral

nucleocapsids Hepatocytes accumulate up to approximately 50 copies of cccDNA per cell (33) Early in infection, the concentration of the envelope protein is insufficient for virion assembly and mature nucleocapsids migrate to the nucleus to restore the cccDNA

by up to 50 copies per cell Ultimately, viral envelope proteins are produced in vast excess over the required amount for virion assembly thereby ensuring the shutdown of cccDNA formation, as additional accumulation of cccDNA may be cytocidal if allowed

to proceed unchecked (34;35)

1.2.2 Hepatitis B virus particle

During HBV infection in humans, virus particles are present in very large quantities in the blood and both infectious and non-infectious particles can be found in the serum of acutely infected individuals The infectious hepatitis B whole virion (Dane particle) is a 42 nm sphere that consists of an envelope and a nucleocapsid (Figure 4) The outer envelope contains large amounts of hepatitis B surface proteins The inner nucleocapsid surrounded by the envelope comprises of 180 to 240 hepatitis B core proteins (HBcAg, 20 kDa polypeptide chain of each) and encloses the HBV DNA molecule as well as at least one hepatitis B polymerase protein (36) Two subviral non-infectious particles, which do not contain hepatitis B core, DNA genome and polymerase, can be found in large amounts in the circulation system of infected individuals These non-infectious particles are called hepatitis B filament and hepatitis B sphere Both have

an average width of 22 nm and are composed of hepatitis B surface proteins The sphere contains SHBs and MHBs, whereas the filament also includes the LHBs These non-infectious particles have the same antigenic site as the infectious complete virion It is

Figure 4 Electron micrograph of HBV The core with an outer envelope is

shown in the left image The right image shows the whole viral particles and the HBs filaments and spheres

(http://www.uct.ac.za/depts/mmi/stnnard/emimages.html)

generally believed that the infectious virions transverse the blood stream without being detected by neutralizing antibodies when there are a large amount of non-infectious particles present (37)

1.2.3 The HBV genome

The HBV genome is one of the smallest human viruses and enclosed in the nucleocapsid of a virion It is a circular, partially double-stranded DNA at 3.2 kb in length One strand of the HBV genome, which is complementary to the viral mRNA and named the minus strand, is full length The other, the plus strand, is always incomplete within the virion The 5’ end of the plus strand is fixed, but the 3’ end is variably situated

in different virus particles (Figure 5) The genome can be made completely double

stranded by viral DNA polymerase if dNTPs are added in vitro (12;38) Unlike other

viruses, the HBV virion contains both DNA and RNA Moreover, some regions of the

packaged genome can be single stranded, double stranded or triple stranded The HBV has a very compact genome structure containing four defined overlapping open reading

Figure 5 HBV genome and open reading frames The numbers on the

genome (0-3221 bp) are based on the adw2 subtype of HBV Transcription of the cccDNA is governed by enhancers I and II, the glucocorticoid response elements (GRE) and the promoters upstream of the mRNA start sites The open reading frames are defined by the first start codon of protein synthesis and the first stop codon Some in-frame internal start codons are also shown (Modified from: http://www-micro.msb.le.ac.uk/3035/HBV.html)

frames (ORFs) They are designated S, C, X, and P The P region overlaps all other ORFs (Figure 5) In addition, HBV encodes more than one protein from one ORF due to the presence of in-phase start codons Therefore, seven proteins can be translated from the four ORFs contained in the HBV genome ORF S encodes the three surface proteins; ORF C codes for hepatitis B e and c proteins; ORF X encodes x protein and ORF P,

which occupies the majority of the genome, codes for the hepatitis B polymerase The HBV genome can actually be read one and half times Furthermore, numerous genetic elements that regulate transcription and determine the polyadenylation site are placed within the coding region (Figure 5)

1.2.4 HBV promoters and signal regions

There are many important promoter and signal regions necessary for viral replication in the HBV genome Transcription of the four ORFs is regulated by four independent promoters (pre-S1, pre-S2, core and x) and two enhancer elements-enhancer

I and II (Table 1 and Figure 2) There are at least four transcripts of different sizes transcribed by the cellular RNA polymerase II As a result of the unique polyadenylation signal TATAAA shortly after the start of the HBc gene, all viral transcripts share a common 3’ end (Figures 2 and 5)

Table 1 Transcription factors (TFs) required for activation of HBV enhancers and promoters

controls transcription of 2.4 kb mRNA that codes for LHBs (liver-specific)

HNF4, SP1, etc regulates transcription of 3.5kb pgRNA that is

template of RT and encodes HBcAg, DNA polymerase

Promoter

x nt 1230-1376 X-PBP controls transcription of 0.7kb mRNA coding for HBx

Three distinct hepatitis B surface proteins are translated using in-phase start codons in ORF S Two promoter regions control the transcription of mRNAs that codes for these proteins, termed the pre-S1 promoter and the pre-S2 promoter The pre-S1 promoter regulates transcription of a 2.4 kb mRNA, which spans the entire ORF S region and codes for LHBs protein This transcription is regulated in a tissue-specific manner, since the pre-S1 promoter requires the liver specific transcription factors HNF1 and HNF3 for activation (39-41) The pre-S2 promoter controls transcription of a 2.1 kb mRNA transcript which codes for the MHBs and SHBs antigens NF-Y, the CCAAT-binding factor, and Sp1 appear to be involved in the activation of the pre-S2 promoter (42;43) The pre-S2 promoter is functionally stronger than the pre-S1 promoter and, as such, an excess of the SHBs protein (or major surface protein) is synthesized over the LHBs and MHBs antigens (44)

The core promoter (CP) plays a very important role in HBV replication and morphogenesis It directs transcription of two classes of 3.5 kb supergenomic RNAs, the longer precore mRNA and an approximately 30 nucleotide shorter pgRNA, which have 5’ heterogeneity The pgRNA codes for the core protein and polymerase and serves as the template for reverse transcription of HBV after encapsidation into core particles (13) The precore mRNA is translated into the precursor of HBeAg Although the CP has not been clearly defined, it is believed to be contained between nt 1591 and 1851 (45) The CP consists of the basic core promoter (BCP) and the upper regulatory region (URR) (Figure

6 a) The URR (nt 1742) contains a negative regulatory element (NRE nt 1636) and a core upstream regulatory sequence (CURS nt 1636-1742) The NRE is located further upstream and is composed of at least three subregions, NRE-α, NRE-β

1613-Figure 6 Organization of the HBV core promoter (a) The CP is a single

regulatory region overlapping the 3’ end of the X ORF and the 5’ end of the precore/core ORF The major functional elements are the upper regulatory region (URR) and the basic core promoter (BCP) The URR consists of a negative regulatory element (NRE) and a core upstream regulatory sequence (CURS) The CURS can be subdivided into unitary sequence motifs that positively regulate BCP activity, namely α, γ and δ, whereas β negatively regulates BCP activity (b) Initiation sites of precore mRNA and pgRNA transcription are shown as horizontal arrows The four AT-rich regions are underlined and the initiators for precore mRNA (pre-C Inr) and pgRNA (pgRNA Inr) are indicated by the rectangular boxes [Kramvis A, 1999 (45)]

and NRE-γ, which synergistically suppress CP activity The positive regulatory element located directly 5’ of the BCP is the CURS which overlaps with enhancer II The CURS can be subdivided into tow domains: CURS A (nt 1636-1730) and B (nt 1704-1743) The CURS can be further subdivided into unitary sequence motifs α (nt 1646-1668), γ (nt 1671-1686), δ (nt 1687-1703) that positively regulate BCP activity and β (nt.1704-

(a)

(b)

been defined as nt 1687-1805, nt 1627-1732, nt 1646-1741 and nt 1646-1741

Enhancer II is composed of two interacting sequence motifs, a 23-bp box α (nt 1668) and a 12-bp box β (nt 1704-1715) (Figure 6 a) A number of transcription factors can bind to the α box The BCP is the minimal essential promoter sequence

1646-accurately mapped from nt 1742 to 1849 The BCP contains cis-acting elements that

independently direct the transcription of supergenomic precore mRNA and pgRNA The BCP has no canonical TATA box (TATAAA) but contains four TATA-like boxes (AT-rich regions) that bind recombinant TATA-binding protein (TBP) (Figure 6 b) Three of the AT-rich regions located 20 to 35 bp 5’ to the precore mRNA start sites are required for the optimal transcription of precore mRNA (TA1, nt 1750-1755, TA2, nt1758-1762, TA3, nt 1771-1775) TA4 (nt1788-1795) functions as both a TBP-binding site for initiation of pgRNA transcription and as the initiator sequence for some of the precore mRNAs (45)

The regulation of CP also involves in a number of transcriptional factors present

in various tissues and cell types that can bind to their cis-acting elements on the CP

during different stages of liver cell differentiation and thus confer the liver cell specificity

of HBV (Figure 7) CCAAT/enhancer binding protein (C/EBP), a liver-specific nuclear factor, binds to multiple sites on the CP to activate the CP at low concentrations and repress it at high concentrations (46;47) The sequence from nt 1718 to 1736 identified

as the target for a liver specific transcription factor-hepatocyte nuclear factor 1 (HNF1) that activates the transcription of several liver specific genes, overlaps with the binding site of another liver-enriched transcription factor HNF3 involved in transcription activation (48;49) In addition, HNF4 binds to two sites on the CP and the one in the BCP

Figure 7 Transcription factor binding sites on the HBV core promoter

Binding sites for rat nuclear liver extract (RNLE), CCAAT/enhancer binding protein (C/EBP), hepatocyte nuclear factor (HNF) 1, HNF3, HNF4, liver enriched factor (LEF), SP1 protein, and TATA-binding protein (TBP) are shown AT-rich regions are shown in grey boxes [Kramvis A, 1999 (45)]

partly overlaps the binding site of an uncharacterized liver enriched factor (LEF) (50-52)

A number of members of the nuclear receptor superfamily of transcription factor including retinoid X receptor (RXR), chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1), peroxisome proliferator-activated receptor (PPAR) and apolipoprotein AI regulatory protein 1 (ARP1) bind to this region (nt 1752-1777), differentially regulating the synthesis of precore mRNA and pgRNA (51;52) (Figure 7) HNF4 specifically down-regulates precore and PPAR only activates pgRNA synthesis, whereas COUP-TF coordinately represses the synthesis of both transcripts The ubiquitous transcription factor, Sp1, specifically increases pgRNA transcription, but not that of precore mRNA, by binding to three sites on CP at nt 1621-1632, nt 1731-1740 and nt 1743-1752 (53)

The X promoter spans from nt 1230 to1376 and controls the transcription of the shortest 0.7 kb RNA from ORF X The expression of X mRNA requires a trans-acting cellular factor that specifically interacts with the X promoter, referred to as the X

promoter-binding protein (x-PBP as in Table 1) This factor binds to a site (nt 1246) that overlaps a cluster of transcription initiation sites for the X mRNA and 3’ end

1225-of enhancer I (54)

Enhancer I spans from nt 970 to 1240 on the HBV genome within ORF P and plays an important regulatory role in the activation of the HBV surface antigen and core promoter elements Enhancer II is situated 600 nucleotides downstream of enhancer I in the ORF X, overlapping the core promoter It has a particularly significant stimulatory effect upon the pgRNA promoter but is also active on the S promoter Both enhancers can activate the heterologous promoters with a significant liver specificity despite their position and orientation (54)

1.2.5 HBV Gene products

1.2.5.1 Hepatitis B surface proteins

ORF S contains three in-phase start sites and codes for a family of hepatitis B surface antigen proteins, namely SHBs, MHBs and LHBs, that make up the viral envelope (Figure 8) These related proteins share a region known as the S domain The major hepatitis B surface antigen (HBsAg) or the SHBs, historically called the Australia antigen, is the smallest protein in this family and is encoded by the S gene The SHBs consists of only the S domain and is highly hydrophobic, containing many tryptophans and 14 cysteines that are cross-linked with each other This peptide carries the major antigenic determinants that allow for subtyping analysis of HBV carriers In the early stage of infection, this protein is produced in the greatest quantities in the infected cells Expression of the SHBs is thought to be inducible by stress in the ER due to the presence

Figure 8 Schematic model of the HBV and HBs particles The SHBs

protein of the viral envelope is identical to the S domain of MHBs and LHBs The MHBs contains a small pre-S2 domain and the LHBs contains a pre-S domain composed of the pre-S2 and pre-S1 sequences The viral envelope encloses a nucleocapsid that encapsidates the 3.2 kb HBV DNA genome, the DNA polymerase (pol), protein kinase C and the heatshock protein hsp90 The filaments consist of the same protein as the virion envelope The spheres contain fewer LHBs [Bircher J., 1999 (55)]

of high levels of LHBs (56)

MHBs consists of the S domain and an additional 55 amino acid of the pre-S2 domain The pre-S2 domain is hydrophilic and does not contain cysteine LHBs is the largest hepatitis B surface protein containing the pre-S1, pre-S2 as well as S domains The pre-S1 domain is believed to be the HBV protein that is involved in liver attachment (57) This domain is also required for envelopment of the core particles by surface proteins (58) Overexpression of LHBs relative to expression of SHBs prevents secretion

of HBs particles and results in ER retention of filaments (59) SHBs is the most abundant polypeptide in all three HBV associated particles, whereas MHBs is a minor component LHBs is more prevalent than MHBs in virions and filaments, but less prevalent in HBs

1.2.5.2 Hepatitis B core and e proteins

The ORF C (precore/core) encodes two related but functionally distinct proteins: hepatitis core protein (HBcAg) and hepatitis e protein (HBeAg) ORF C has 212 or 214 codons, depending on the genotype of the virus The translation of the two proteins starts

at the first and the in-phase second start codon (29 codons downstream of the first AUG)

Precore/core gene

183aa HBcAg

29aa 183aa

Precore protein (precursor of HBeAg) 10aa 150aa

HBeAg

Figure 9 Expression of core and e proteins from the precore/core gene

The HBcAg is translated from pgRNA using the AUG codon at nucleotide

1901 The precore protein is translated from precore mRNA using the AUG codon at nucleotide 1814 Removal of 19 aa residues at the amino terminus and around 34 aa at the carboxyl terminus generates HBeAg (60;61)

respectively Thus, HBcAg and HBeAg share the primary 150 aa sequence (Figure 9) However, HBcAg is encoded by the shorter species of 3.5 kb transcripts, the pgRNA, and the resulting protein is located in the cytoplasm and the nuclei of the cells It self-assembles into virus capsids and is exported from the cells only as part of a virus particle The 25 kDa precore protein (precursor of HBeAg) is encoded by the larger 3.5 kb species, the precore mRNA, and contains an additional short sequence at the N-terminus ATG ATG TAG

1814 1901 2456

that directs the precore protein to the endoplasmic reticulum (ER) as a signal sequence The mature HBeAg has 10 additional amino acids at the amino terminus but lacks 34 highly basic amino acid residues that are present at the C-terminus of the HBc protein Thus, HBc readily dimerizes to form core particles and HBe does not The different physical states of these proteins are also reflected by their unique antigenicities The major HBc epitope appears to be a conformational epitope which requires HBc aggregation If the aggregates are denatured, HBc antigenicity disappears (62) On the other hand, there are at least two different HBe epitopes (63;64)

HBcAg contains many hydrophilic and charged amino acids but no lipid glycan This 183 or 185 aa protein is synthesized in the cytosol of infected hepatocytes It is the major component of the nucleocapsid shell packaging the HBV genome and viral polymerase The region associated with HBc nucleocapsid assembly is located within the first 149 aa (65) The crystal structure of the nucleocapsid has recently been resolved HBc is largely a helical protein containing five alpha helices It has been shown that the bacterially expressed core protein assembles to give two different sized shells, composed

of 180 or 240 subunits arranged with T=3 or T=4 icosahedral symmetry Two monomers

of HBc associate to form a compact dimer in which the two α-helical hairpins pack to form a four-helix bundle Dimer association is required for nucleocapsid formation and appears to involve residues Tyr 132, Arg 12, Pro 129 and Ile 139 (66) The C-terminus of HBc protein is extremely rich in arginine, serine and proline residues that interact with the viral nucleic acid The arginine-rich region starts at aa 150 and contains four arginine clusters that are involved in nucleic acid packaging, but are dispensable for particle self-assembly After assembly into core particles and encapsidation of the viral genome, the

hepatitis B surface proteins form the viral envelope, completing construction of the virus Regions within the carboxyl-terminal part of the HBc subunits are essential for genome maturation (28;67)

The entire ORF C codes for the 25 kDa precursor of HBeAg (p25) which contains

29 additional aa at the amino terminal end of the core protein This additional 29 aa domain contains a hydrophobic α helix that serves as a signal sequence which directs the precursor to the ER (68) Nineteen of the 29 extra amino acids of most p25 precore proteins are cotranslationally cleaved and the resulting 22 kDa protein (p22) is translocated into the lumen of the ER The remaining 10 additional amino acids prevent assembly of HBe protein to core particles As the translocation of the HBe protein across the ER membrane is an inefficient process, a significant amount of HBe protein is released back to the cytosol (69) After the HBe protein has reached the Golgi compartment, the arginine-rich carboxyl terminus (about 34 amino acids) is cleaved off

by a cellular aspartyl-like protease at multiple sites resulting in a 16-20 kDa protein that gives rise to the mature HBe protein- HBeAg The soluble HBeAg is then secreted in a monomeric form into the blood of patients (70)

The function of HBeAg in the life cycle of HBV is still enigmatic Even though a role for infectivity or viral multiplication has been excluded (71-73), conservation of this antigen among all the members of the hepadnavirus family suggests that it has an important role in the life cycle of hepadnavirus It has been proposed that the precore protein or the processed shorter p22 form may play a role in virion morphogenesis (60;68) There is evidence to suggest that HBeAg may function as a circulating protein that blocks cytotoxic T-cell activity against HBV core associated epitopes (74) It has

been demonstrated that HBeAg determinants are expressed on the surface of infected hepatocytes and present HBeAg and HBcAg epitopes in the context of HLA class I molecules to the host immune response (75)

The HBeAg is a non-particulate protein in the serum of HBV infected individuals,correlating with high levels of viremia It has been used as an indicator of severity and infectivity of hepatitis B HBeAg seroconversion (SC) from HBeAg positive to antibody

to HBeAg (anti-HBe) poistive is normally accompanied by a viral load drop and normalization of alanine aminotransferase (ALT) to signify a fall in viral replication and infectivity

1.2.5.3 Hepatitis B polymerase protein

ORF P, which is the largest ORF in the HBV genome, encodes a 90 kDa hepatitis

B polymerase protein This protein has RNA and DNA dependent polymerase activity and plays an important role in reverse transcription and pgRNA encapsidation The polymerase protein is only packaged within the core particle together with the pgRNA Hepatitis B polymerase protein contains four clearly distinguishable domains The amino-terminal domain of this protein links to the 5’ end of the minus-strand of viral DNA This domain is necessary for priming of minus-DNA synthesis, and thus named primase The next domain does not have any specific function and appears to act as a spacer The third domain occupies approximately 40% of the protein and provides the RNA and DNA dependant polymerase activity Evidence suggests that the polymerase/reverse transcriptase activity of hepatitis B polymerase requires the presence

of metal ions and the stem-loop (76;77) The last carboxyl-terminal domain of hepatitis B polymerase has RNase H activity that cleaves the RNA present in RNA/DNA hybrids

1.2.5.4 Hepatitis B x protein

The smallest ORF X codes for the 16.5 kDa hepatitis B x protein (HBx) whose function is poorly understood HBx is highly conserved between human, woodchuck and ground squirrel hepadnaviruses, although it is absent in the avian hepadnaviruses HBx protein is not a structural component of either the virion or nucleocapsid particles and studies suggest that HBx predominantly localizes to the cytoplasm (78) A variety of functions have been ascribed to this protein Some suggest that HBx protein activates transcription of many genes (79;80) There is also evidence that HBx is required for virus

expression in vivo (81) One of the most significant effects of HBx may be its

tumorigenic activity Overexpression of HBx in hepatocyte culture and transgenic mice results in a tumorigenic phenotype (82;83) Some evidence shows that HBx is involved in regulating Sp1 mediated transcription of an insulin-like growth factor II (IGF-II) (84) Furthermore, an interaction with a tumor suppressor protein p53 has been postulated (85)

1.2.6 HBV genotype and HBsAg subtype

The subtypes of HBV were originally defined by antibodies arised to the SHBs Antigenic determinants present on all known HBs isolates were considered as

determinant a Four other major determinants (two pairs of mutually exclusive determinants) are d or y and w or r Determinant d has a lysine at position aa 122, y an arginine Similarly, determinant w has a lysine at aa 160, r an arginine There are four

sub-serotypes (adw, ayw, adr and ayr) based on the subtypes of HBsAg With the description

of four subdeterminants of a that was later redefined as subdeterminants of w1-4 (Table 2) and the identification of the q determinant, the number of subtypes has been increased

Table 2 Amino acid residues specifying determinants of HBsAg

122

127

160

Lys Arg Pro Thr Leu/Ile Lys Arg

w1 reactivity also requires Arg 122, Phe 134 and/or Ala 159

to nine These are ayw1, ayw2, ayw3, ayw4, ayr, adw2, adw4, adrq-and adrq+ (86-88)

With the emergence of the complete HBV genomes, six genotypes from A to F were identified based on more than 8% of differences of the genome and recently, new genotypes G and H have also been identified (89-92) The F genotype is the most divergent, with a divergence of 14% In general, genotype A occurs commonly in Northwestern Europe and sub-Saharan Africa, genotypes B and C in the Far East and Asia, genotype D is most widespread in the Mediterranean area and the Near East, genotype E is found only in West Africa, genotype F in the aboriginal populations

of the Americas, genotype G in France and North America and the new Amerindian genotype H has been identified from Central America The genotype of HBV genomes in Singaporean hepatitis B patients remains to be determined The relationship between genotypes, HBsAg subtypes and geographical distribution are summarized in Table 3

Table 3 Geographical distribution of HBV genotypes and subtypes

adrq- ayr ayw2 ayw3 ayw4 adw4q- adw2 adw4

Northwestern Europe Central Africa Indonesia, China Vietnam East Asia Korea, China, Japan Polynesia Vietnam Mediterranean area India West Africa American natives, Polynesia France, north America Central America

1.3 HBV: the disease

Viral hepatitis is an inflammation of the liver caused by the hepatitis viruses Five different hepatitis viruses have been identified Two viruses, hepatitis A and E, only cause acute hepatitis whereas hepatitis B and C viruses cause both acute and chronic infections Delta virus is a viroid that can only infect patients who are already infected with HBV Recently, a blood-transmissible virus has been identified in certain cases of chronic hepatitis and is termed GB-virus C or hepatitis G virus (93) However, evidence

is accumulating that HGV does not replicate in the liver and does not cause hepatitis

HBV infection, which is caused by HBV, a small enveloped DNA virus that infects the liver, causing hepatocellular necrosis and inflammation remains an important worldwide disease HBV infection induces a spectrum of clinical manifestations, ranging

from mild, unapparent disease to fulminant hepatitis, severe chronic liver disease and cirrhosis The virus also has been clearly implicated in the development of primary hepatocellular carcinoma (HCC), one of the eight most common cancers in the world

1.3.1 Prevalence

The prevalence of HBV infection varies in different geographical areas In prevalence areas such as the United States, Western Europe, Australia and New Zealand, the HBsAg carrier rate is approximately 0.1 to 2% In intermediate prevalence areas like the Mediterranean countries, Japan and India, the carrier rate is approximately 3 to 5%, while in high-prevalence areas such as Southeast Asia, China and sub-Saharan Africa, the carrier rate is 10 to 20% In Singapore, the overall HBsAg carrier rate in the generally population is estimated to be approximately 4.1% and the incidence rate of primary liver cancer ranks as the fourth commonest cancer for male in 1999 (94)

low-Hepatitis B infection is a global public health problem and it is estimated that there are more than 300 million chronic HBV carriers In adults, 90-95% of cases resolve spontaneously with varying degrees of severity of acute hepatitis B The remaining 5-10% of adults develops chronic hepatitis B However, in children infected between the ages of 1 and 5 years, 29-40% of them have chronic hepatitis In contrast, the chronicity can be as high as 90% in infants born to HBeAg positive mothers This mother-to-infant vertical transmission perpetuates the chronic infection from generation to generation (95) The ratio of males to females with hepatitis B infection is usually between 1.5:1 and 2:1

1.3.2 Transmission

Hepatitis B is not as highly contagious as, for instance, hepatitis A However, the presence of large amounts of virus in the blood and secretions of infected persons, which persists over a long time, means that even an isolated, single and minute exposure can transmit this infection The spread is facilitated by the fact that many chronic HBsAg carriers are asymptomatic and unaware of their infection and potential infectivity

Transmission of HBV is largely from the parenteral route The source of most HBV infections is exposure to blood from HBV-infected individuals In the 1960s, the risk of hepatitis B infection from commercial blood transfusions was as high as 50% Currently, the risk of HBV transmission by transfusion of screened blood is very small (96) Percutaneous inoculation of blood or body fluid plays a major role in the transmission of hepatitis B infection Needle sharing by intravenous drug users, reusing

of contaminated needles for tattoos, acupuncture and ear piercing all facilitate transmission of hepatitis B Sexual spread of hepatitis B among homosexuals as well as heterosexuals also appears to occur Hepatitis B can also be spread by intimate contact that is not apparently sexual or parenteral The most important mode of transmission of hepatitis B is perinatal spread from HBV-infected mothers (mother to child) In high-prevalence areas, such as Hong Kong and China, perinatal transmission accounts for 40

to 50% of chronic HBV infection (95;97) In areas of low endemicity, mother-child or

child-child transmission is rare

1.3.3 Clinical manifestation and diagnosis

The clinical presentation of HBV infection has a very broad spectrum, ranging from asymptomatic patients that have no detectable evidence of liver disease, to severely ill patients with jaundice, edema, ascites, upper gastrointestinal bleeding and other severe presentations

Acute hepatitis B is an active inflammation and necrosis of the liver that occurs in association with a usually transient HBV infection The diagnosis of acute hepatitis B is based on the detection of HBsAg and IgM anti-HBc in the serum of a patient with clinical and serum biochemical evidence of acute hepatitis During the initial phase of infection, markers of HBV replication, including HBeAg and HBV DNA, are present Recovery is accompanied by the disappearance of HBV DNA, HBeAg to anti-HBe SC and subsequent HBsAg to anti-HBs SC Rarely, patients may have entered the window period

in which only IgM anti-HBc can be detected in the serum at the time of presentation As such IgM anti-HBc is the sole marker of acute HBV infection in these patients This situation is more common in patients with fulminant hepatitis B, in which virus clearance tends to be more rapid

The incubation period of acute HBV infection is from 1 to 4 months During the prodromal period, a serum sickness-like syndrome may develop This is followed by the insidious onset of constitutional syndromes including malaise, anorexia, nausea, occasionally vomiting, low-grade fever, myalgia, and easy fatigability Some patients may experience intermittent, mild-to-moderate right quadrant or midepigastric pain In patients with icteric hepatitis, jaundice usually begins within 10 days of the onset of constitutional symptoms Clinical symptoms and jaundice usually disappear after 1 to 3

months, but some patients may have persistent fatigue, even after the aminotransferase levels have returned to normal

The disease generally lasts 1 to 6 weeks, but may be prolonged and can be fulminant Not all patients with acute hepatitis B infection develop clinically apparent acute hepatitis B It is estimated that 65% of acutely infected adults do not manifest an overt illness This group of patients shows a subclinical infection producing antibody and seemingly permanent immunity (Figure 10) Recovery is the most likely outcome for another 25% of patients who have acute hepatitis, and only 1% of them develop fulminant hepatitis with high mortality (98) The remaining 10% of acutely infected individuals develop chronic hepatitis B infection, which is more important in the epidemiology and worldwide significance of HBV since these individuals may progress

to cirrhosis, primary hepatocellular carcinoma (HCC), or both (99)

Chronic hepatitis B is defined as HBV infection for more than six months and usually lasting many years The diagnosis of chronic HBV is based on the detection of HBsAg in the serum for 6 months or more It is characterized by persistent HBV infection associated with inflammation and hepatocellular necrosis, or without any evidence of liver disease Many patients with chronic HBV infection are asymptomatic, whereas others have nonspecific symptoms such as fatigue Occasionally, mild right upper quadrant or midepigastric pain may be present Patients with chronic HBV infection may experience exacerbations that may be asymptomatic or mimic acute hepatitis with fatigue, anorexia, nausea, and jaundice, and in rare instances, progress to hepatic decompensation Some chronic patients eventually show normalization of liver

enzymes and clear the infection with the development of anti-HBs; others do not resolve the infection and ultimately develop cirrhosis, liver failure and HCC (Figure 10)

Figure 10 Outcome of hepatitis B virus infection in adults [Hoofnagle J

serum alanine aminotransferase (ALT) levels, and minimal histological activities, which imply the lack of or a very weak immune response against the infected hepatocytes Once the tolerant effect is lost during the course of chronic HBV infection, patients may enter the second phase- the immunoactive phase, which is associated with a decrease in HBV DNA concentration and increased ALT levels and histological activity, reflecting immune-mediated lysis of infected hepatocytes The second phase lasts from months to years The third nonreplicative (or low replicative) phase which is also referred to as the inactive HBsAg carrier states occurs after SC from HBeAg to anti-HBe This phase is usually preceded by a marked reduction of serum HBV DNA to undetectable levels by hybridization techniques, followed by normalization of ALT levels and resolution of liver necroinflammation The third phase may last for a lifetime, but a proportion of patients may undergo subsequent spontaneous or immunosuppresion-induced reactivation of HBV replication with reappearance of high levels of HBV DNA, with or without HBeAg

SC and a rise in ALT levels Patients who have SC from HBsAg to anti-HBs are diagnosed as have resolved hepatitis B (100)

Chronic HBV infection is found to be an important cause of morbidity and mortality in follow-up studies of chronic hepatitis B patients and the studies in HBV endemic areas Progression from chronic hepatitis to cirrhosis and from compensated cirrhosis to hepatic decompensation and HCC has been estimated to be 12 to 20%, 20 to 23% and 6 to 15% at 5 years, respectively (101-104) The life-time risk of a liver-related death is estimated to be 40 to 50% for men and 15% for women among Chinese patients with chronic HBV infection (105) Approximately one million people die each year from

complications of the disease that include cirrhosis, liver failure and HCC, making chronic hepatitis B one of the ten most common causes of death

1.4 Therapeutic treatment of hepatitis B

Progression to cirrhosis and end-stage liver diseases, with high risk of development of HCC, are the main complications for chronic hepatitis B patients These progressions tend to occur in patients with active viral replication, reflected usually by the presence in serum of detectable HBeAg and HBV DNA Furthermore, HBV can be transmitted from chronic hepatitis B patients or ‘healthy’ carriers Therefore, the aim for the treatment of hepatitis B is to clear the HBV from patients and thereby prevent transmission to other people and the progression to the advanced stages of liver disease

A successful response to therapy is defined as the sustained clearance of HBeAg (HBeAg SC to anti-HBe) and HBV DNA (by hybridization assays) with the eventual loss

of HBsAg

An efficacious treatment against HBV infection has still not been found till now Several potentially effective agents with different mechanisms of action have entered clinical trial or clinical practice Among these, interferon alpha and lamivudine have been the most widely studied and their use has been licensed in many countries (106)

1.4.1 Interferon therapy

Interferons are cytokines with immunomodulatory, antiproliferative and antiviral properties Interferon alpha (IFN-α) belongs to the type I interferon protein family, which includes many closely related glycoproteins released by virally infected cells There are

at least thirteen different IFN-α subtypes, one IFN-β and one IFN-ω subtype and it is believed that these different subtypes of IFN have similar functions

IFN-α has two main functions that are thought to be important for treatment of chronic hepatitis B The first function is antiviral, with inhibition of viral protein synthesis IFN-α induces an increase in the intracellular levels of 2’-5’-oligoadenylate synthesis which can activate ribonucleases and thus cleave viral mRNA The second main function of IFN-α is its immunomodulatory effects, including the increase in the major histocompatibility complex (MHC) class I display of the hepatocyte and an increase in natural killer cell activity Thus, the immune system can efficiently recognize infected hepatocytes, which can be destroyed by cytotoxic T cell recognition of MHC class I display (107)

IFN-α has been used for treatment of chronic HBV since early 1970 and the first

successful use was reported by Greenberg et al in 1976 (108) IFN-α, which was licensed

in 1992, is the first approved treatment for chronic hepatitis B infection in most countries The rate of successful response to this treatment is 30% to 40% in chronic hepatitis B patients who are treated subcutaneously at a daily dose of 5 million units or three times a week at 10 million units (109;110) The relapse rate is around 5% to 10% Long-term observation of IFN treated patients compared to untreated controls has been documented and an improvement in clinical outcome, including prolonged survival, reduced fatality, and reduced frequency of HCC has been observed (111) Successful interferon therapy is more likely in patients with low level HBV DNA and substantial elevation of aminotransferase activity However, those with underlying disease or immunosuppression are unlikely to respond to interferon therapy Interferon treatment may be dangerous in

patients with decompensated liver function In patients with cirrhosis, interferon may lead

to decompensation

IFN treatment is usually associated with side effects, especially flu-like symptoms, depression, neutropenia, and thrombocytopenia These are normally tolerable but sometimes require dose modification

1.4.2 Lamivudine therapy

Nucleoside analogues, such as lamivudine and famciclovir, lack the equivalent of the 3’ hydroxyl group on the (deoxy) ribose sugar and act as growing viral DNA chain terminators All the nucleoside analogues have to be phosphorylated intracellularly for antiviral activity before they can act as competitive inhibitors and they are very effective

in inhibiting active viral replication and reducing viral load at low intracellular drug concentrations (112;113)

Lamivudine, a cytosine analog, which has been approved as antiviral therapy for chronic hepatitis B, is effective in HBV DNA suppression, ALT normalization and improving liver histology in both HBeAg positive and HBeAg negative patients and is able to interrupt hepatic decompensation successfully and safely (114) In addition, Lamivudine is well tolerated with a negligible rate of significant adverse side effects

A major problem with lamivudine therapy is the development of drug-resistant mutations A point mutation in the YMDD (Tyr-Met-Asp-Asp) motif that codes for the catalytic site of the DNA polymerase of the HBV genome has been found in lamivudine resistant clinical isolates The frequency of YMDD variants ranges between 15% and

32% after a year of therapy, increasing to 67% after 4 years of therapy The occurrence of these mutants may be associated with more severe liver damage (115)

Thymosin-α1, a thymic extract, is a 28 aa peptide presumed to stimulate cytolytic

T cells, promote interferon α, γ, IL-2 and IL-3 production by normal lymphocytes and increase lymphocyte IL-2 receptor expression (116;117) This treatment showed early promise in a small clinical trial, but the efficacy was not supported by the results of a large multicenter, randomized, placebo-controlled clinical trial A recent trial in Asian patients suggested that thymosin-α1 has benefits (118)

1.4.4 Other alternative therapies

Since lamivudine and interferon treatment have different mechanisms of function, combination of these two agents may have synergistic effects However, some recent studies show that combination therapy was no better in complete response rate than monotherapy (119;120)

Other nucleoside analogues with early promise including famciclovir, lobucavir, entecavir, emtricitabine (FTC) and adeforvir dipovoxil are being studied currently Famciclovir, a deoxyguanosine nucleotide analog, has been reported to inhibit the replication of HBV but with weaker action than lamivudine (121) Adeforvir dipovoxil (an acyclic analogue of dAMP) administration has resulted in a rapid decrease in serum HBV DNA in phase I and II studies but with no resistance even after 52 weeks continuous treatment (115)

1.5 Hepatitis B viral mutants

Antiviral therapy and/or host-immune response may drive the selection of HBV mutants during hepatitis B virus infection Moreover, mutations of HBV may affect some part of the HBV life cycle and change the outcome of the natural course of HBV infection Therefore, studies of viral mutations over time within the overall viral population in relation to host-virus interactions are extremely important for understanding the biological and pathogenic role of the HBV mutants

Viral DNA and especially RNA genomes are inherently variable due to errors introduced during replication These errors are in the range from 10-3 to 10-5 substitutions per nucleotide per replication cycle for RNA viruses This is due to the lack of proofreading functions of RNA polymerase and reverse transcriptase In contrast, there is only about 10-8 substitutions per nucleotide per replication cycle for DNA viruses (122) HBV, in spite of a high replication efficiency that produces 1011 copies of circulating virus daily, shows a mutational rate of lower than 2 × 10-4 base substitutions per site per year, which is four orders of magnitude greater than that of other DNA viruses, because they replicate asymmetrically through reverse transcription of an RNA intermediate However, the compact organization of the HBV genome, with multiple overlapping ORFs, reduces the number of viable mutants and the rate of their production Thus, the mutation rate of HBV is 100 to 1000 times lower than that of RNA viruses As mutations occur along the HBV genome, selection pressure would permit the emergence of HBV mutants that might be ‘fitter’ than wild type virus (122)

The basic classification of genomic mutations identifies three major categories: