Hepatitis b virus gene mutations associated with HBeAg seroconversion 2

Chapter 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 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 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)] 1.2.1.3 HBV gene translation HBV usually uses the free ribosomes for synthesis of nucleocapsid proteins and ER-bound ribosomes for envelope protein synthesis The 3.5 kb supergenomic RNA transcripts have two subsets that differ by approximately 30 nucleotides at their 5’ end Only the longer subset contains the start codon of precore sequence and encodes for the precursor of hepatitis B e (HBe) protein Core (HBc) proteins and polymerase proteins are translated from the shorter subset that lacks the first start codon of precore sequence The translation of core protein starts at the second in-phase start codon However, the mechanism of polymerase translation is still unclear 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 RNAbinding ability is essential for pregenomic RNA encapsidation (28) 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) 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 10 Cd nn oo o A1 14 F2 23 F S mle ap C n n lo e o C-I /2 40 C-I 10 /2 C-I 10 /2 C-I 10 /2 C-I 10 /2 C-I 10 /2 C-I 10 /2 H Cd nn oo o A1 14 F2 23 S mle ap C n n lo e o C-III 80 /2 C-III 20 /2 C-III 30 /2 C-III 20 /2 C-III 10 /2 C-III 10 /2 C-III 10 /2 C-III 10 /2 C-III 10 /2 Y E K K A G K * 3 5 11 12 11 10 18 0 9 M I S D Y L E V G R P Q P G H G M R S R L R * * L T I * S W E E N * L S R V G K L I F * V * G E S S Cd nn oo o A1 14 F2 23 H C L S mle ap C n n lo e o C-V /2 20 C-V 10 /2 Y C-V 10 /2 C-V 10 /2 C-V 10 /2 C-V 10 /2 C-V 10 /2 C-V 10 /2 C-V 10 /2 T A * Codon no AF121243 Sample S2-III S2-III S2-III S2-III S2-III S2-III S2-III S2-III S2-III S2-III S2-III S2-III S2-III S2-III S2-III S2-III S2-III Codon no AF121243 Sample S2-IV S2-IV S2-IV S2-IV S2-IV S2-IV S2-IV S2-IV S2-IV S2-IV S2-IV S2-IV S2-IV Codon no AF121243 Sample S2-V S2-V S2-V S2-V S2-V S2-V S2-V S2-V S2-V S2-V S2-V S2-V S2-V S2-V S2-V S2-V S2-V A G A G H V - - - - - - - 19 14 24 28 29 21 22 5 0 1 R P S R R E Q C G L R P G W D H G L P D G N N V 37 E 57 R 58 D E Q 20 S 22 L 28 W 33 D 42 V A P L L T * P M F H L 10 I L L L L * * * * * * * * G T 28 W 35 Y L Q F P P * * * * * * * V G 13 S 28 W 33 D 34 P L Clone no 2/25 1/25 2/25 1/25 2/25 2/25 1/25 3/25 11/25 Clone no 2/22 1/22 1/22 1/22 5/22 1/22 1/22 1/22 1/22 4/22 1/22 1/22 1/22 1/22 55 S G P C M 52 F H W S S T L 50 S 56 I 65 A V V V V V V V V V S L R P C L 35 Y 41 S 48 L 56 I 71 L V V V V V V F S T T M H C 28 W 33 D T H 56 I C * V W F 67 Y 81 H 89 L 92 G 94 101 107 109 113 114 127 142 150 163 164 182 210 L V D A L V R E S P P G S D D D D D N R Q I K C 89 L 92 G 94 L Q S 89 L N V V D D D V V V D M D A P V * W W I I H H Q Q Q Q Q Q Q Q Q Q * D K * * * * D D K K L L H H 187 R - C P P P C Q Q K K C P P L L A A I C C C C T E P P P P P P P P P V V V G M D G * 92 G 94 L 96 103 109 113 154 164 210 N S A L W P S I * F L V D V G D C S Q G P P P C 96 115 141 164 182 197 210 N V R P G S S V Q * G Q Q Q Q Q Q Q Q Q Q Q K D D D T T G * 86 Q D D V V V * T T 67 Y Q * V L 67 Y A F C T L F V V V V V V V V V V V V V V F V N N L Q S 56 I V L * G V T T I I N N V Y H H H F * * * A G G G S T E Q Q Q Q Q Q K A G P P P G G L Q 92 139 140 164 196 203 210 G F G P R R S R V V L L L L L L V V V V R S E V V V V H V V V * * L L V V Q Q Q Q G Q Q L P * * * V D D Q Q P * 204 R P G L 10 I 28 W 29 G 34 P 39 G 46 S 61 D 83 A 89 L 92 G 117 Y 139 F 159 P 160 A 177 V 196 R 204 R 206 Q 207 S 208 R 211 Q 212 C D T T C G A P G P Y T E H 29 G D S G S * * * * R D D D D T T T T T R E L T H V V C V M L L H L G 34 P 60 L 61 D 72 E 74 P 78 S 92 G 121 N 159 P 160 A 167 P 177 V 191 S 205 S 206 Q 212 C A T S G K R * K K K S G P P T T K G D D N L G N V 143 T 144 V 145 L 160 A 166 A 179 R K L * * * * * * * * D T Q Clone no 1/21 2/21 1/21 1/21 1/21 1/21 1/21 1/21 1/21 1/21 1/21 2/21 3/21 1/21 1/21 1/21 1/21 184 S L N 28 W Clone no 1/20 1/20 1/20 1/20 1/20 8/20 1/20 1/20 1/20 1/20 1/20 1/20 1/20 84 L H L L L L L L L H Q Clone no 3/20 1/20 1/20 1/20 1/20 1/20 1/20 1/20 1/20 1/20 1/20 2/20 1/20 1/20 1/20 1/20 1/20 39 G K Clone no 15/20 1/20 1/20 1/20 2/20 Clone no 1/20 3/20 2/20 1/20 1/20 5/20 1/20 1/20 1/20 1/20 1/20 1/20 1/20 Codon no AF121243 Sample S1-V S1-V S1-V S1-V S1-V S1-V S1-V S1-V S1-V S1-V S1-V S1-V S1-V S1-V D A M Codon no AF121243 Sample S1-IV S1-IV S1-IV S1-IV S1-IV S1-IV S1-IV S1-IV S1-IV 13 12 19 13 15 18 19 21 22 4 7 1 N G E V V T V R Q C S Clone no 1/22 3/22 1/22 3/22 2/22 1/22 1/22 1/22 1/22 2/22 1/22 1/22 1/22 1/22 1/22 1/22 Codon no AF121243 Sample S1-III S1-III S1-III S1-III S1-III S1-III S1-III S1-III S1-III S1-III S1-III S1-III S1-III 4 12 14 29 S V E R E E Cd nn oo o 8 A1 14 F2 23 P S H R S mle ap C n n lo e o C-IV /2 20 C-IV 10 /2 P C-IV 10 /2 R C-IV 10 /2 C-IV 10 /2 C-IV 10 /2 C-IV 10 /2 C-IV 10 /2 S C-IV 10 /2 - Codon no AF121243 Sample S2-I S2-I S2-I S2-I S2-I Codon no AF121243 Sample S1-I S1-I S1-I S1-I S1-I S1-I S1-I S1-I S1-I S1-I S1-I S1-I S1-I S1-I S1-I S1-I 13 S 26 W P N L D A S T 28 W 33 D 41 S 53 F 56 I 58 D L E G T T T T T * * * * * * * * * * G T V V V V V V 96 N 112 E 115 V 116 S 120 V 136 C 180 R 182 G 184 S 185 P 186 R - 205 S 209 E T T * A T T T T T F F T G 110 S R W T G 103 S T T T T T T T T T V T T T T T T 78 S T T T T T T N N N N N N N N D D D D D D D D N N N N N N N N T T T T T T T T A V A K K D D D D D D D D G G G G G G G G R R R R R R R R S S S S S S S S P P P P P P P P R R R R R R R R R R R R R R R R 217 * C o no od n A 12 F2 W Sm a ple C n no lo e S -I /2 62 * S -I 1/22 * S -I 1/22 * S -I 1/22 * S -I 1/22 * S -I 1/22 * S -I 1/22 * 64 84 16 57 13 I S A S L G T P E V V V V V V V I I F T C o no od n 1 21 A 12 F2 M V K W P Sm a ple C n no lo e S -III 6/22 * S -III 1/22 L * S -III 3/22 * S -III 1/22 * S -III 2/22 * S -III 1/22 * S -III 1/22 * S -III 1/22 I * S -III 1/22 * S -III 1/22 * S -III 1/22 N * L S -III 3/22 * C o no od n A 12 F2 Sm a ple C n no lo e S -IV 1/22 S -IV 1/22 S -IV 1/22 S -IV 1/22 S -IV /2 12 S -IV 1/22 S -IV 1/22 S -IV 1/22 S -IV 2/22 S -IV 1/22 S -IV 1/22 F V V V V V V V A A Q Q Q Q Q Q D Q R V V V V V V V V V V V V P P V * G L V V I V I V V V V V V V V H S * * * * T Clone no 11/23 1/23 1/23 8/23 1/23 1/23 Codon no AF121243 Sample S5-V S5-V S5-V S5-V S5-V S5-V S5-V S5-V S5-V S5-V S5-V S5-V S5-V S5-V S5-V Clone no 1/21 1/21 7/21 1/21 1/21 1/21 1/21 1/21 1/21 1/21 1/21 1/21 1/21 1/21 1/21 H L P P H G G G 29 G 34 P 48 L * * * * * * * L L L L L L L N V V V V V 51 D 64 S V V V V V 67 Y Q Q A Q Q Q G 68 R 77 C * * * * * * * * * * S S L L * * R R 48 L 58 D 71 L C C C I I V * * * D D D * * * W W W L 57 R 80 H 93 E 95 M Y P T G V V I Y L 13 S 20 S P S F F F F F F S S S 68 R 71 L 80 H N N R C A T S D A Q V R * S S A L K M 28 38 39 42 45 54 57 61 70 72 86 160 164 179 181 191 192 202 213 W F G V L P R D A E Q A P R R S P R P S S * * * * * * * * * * * T Q Q C * - P R R L - - - K Q S T G Q G Y Y L L I 28 42 43 56 79 91 106 108 113 142 144 164 170 174 181 200 205 W V E I P W E P L E V P S E R P S A S S S S * * * * C no lone 14/22 1/22 1/22 2/22 1/22 1/22 1/22 1/22 V V V Q Q Q * D D D D K H V I S S L L K K R R G * F I I F F F F F F F F F F L V T T T T T T T T Q * D D D D K P S F K T Q G K G E R L 28 35 42 43 80 127 138 168 208 209 212 213 W Y V E H R T I R E C H A A Y R T G A P S C * A * * * * * * * * * * I I * * * 64 S * * 14 30 38 89 94 115 118 157 165 196 208 C M F L L V V T N R R 98 100 103 107 112 113 116 127 A W S D E L S R G S S C no lone 2/21 3/21 1/21 2/21 1/21 2/21 1/21 2/21 1/21 6/21 I 77 105 116 118 127 C L S V R N H H V V F F P L L Q K I I I I G 63 A S F C odon no AF121243 Sam ple S4-V S4-V S4-V S4-V S4-V S4-V S4-V S4-V T G G G P P L L L L L L L P T 20 S L D T S Q Q R Q R R R R R R R C no lone 1/23 1/23 1/23 5/23 5/23 1/23 1/23 1/23 1/23 1/23 2/23 1/23 1/23 1/23 C odon no AF121243 Sam ple S4-IV S4-IV S4-IV S4-IV S4-IV S4-IV S4-IV S4-IV S4-IV S4-IV G D 69 93 14 16 19 2 25 59 84 08 13 I D E L G E K E L P P V S R S R W * L Clone no 1/22 1/22 1/22 1/22 4/22 1/22 5/22 1/22 1/22 1/22 1/22 1/22 1/22 1/22 1/22 N L G 28 W Codon no AF121243 Sample S5-IV S5-IV S5-IV S5-IV S5-IV S5-IV S5-IV S5-IV S5-IV S5-IV S5-IV S5-IV S5-IV S5-IV S5-IV A R C o no od n 19 52 A 12 F2 M L A W F F S Sm a ple C n no lo e S -V 3/21 S -V 2/21 V S -V 2/21 S -V 1/21 S -V 3/21 S -V 1/21 S -V 1/21 S -V 3/21 * S -V 1/21 L * S S -V 2/21 * S T S -V 1/21 * S -V 1/21 P * Codon no AF121243 Sample S5-III S5-III S5-III S5-III S5-III S5-III A R V L Clone no 1/22 8/22 1/22 1/22 1/22 1/22 2/22 1/22 1/22 1/22 1/22 1/22 2/3 V Q Q Q Q Q Q Q Q Q Q Q G C no lone 14/21 2/21 1/21 1/21 1/21 1/21 1/21 C odon no AF121243 Sam ple S4-III S4-III S4-III S4-III S4-III S4-III S4-III S4-III S4-III S4-III S4-III S4-III S4-III S4-III L 56 6 83 95 10 16 43 I L Y S A L G M T D Q T P R 28 3 43 6 75 10 13 4 9 32 70 I W P G E L Y E E L D L F H P S V Codon no AF121243 Sample S5-I S5-I S5-I S5-I S5-I S5-I S5-I S5-I S5-I S5-I S5-I S5-I S5-I C odon no AF121243 Sam ple S4-I S4-I S4-I S4-I S4-I S4-I S4-I 95 116 130 148 151 152 154 157 163 165 178 189 M S L L F G W T P N V T P C no odon A F121243 S ple am N 1-I N 1-I N 1-I N 1-I C no lone 17/20 1/20 1/20 1/20 26 48 71 117 137 W L L Y L C no odon A F121243 S ple am N 1-II N 1-II N 1-II C no lone 17/21 2/21 2/21 S F * M C F C Q V I I P R Q S M G A A C C W W L L L * E E L L L L L L L * G A S L L Y H H M M T S S * * H H P P G G L L G G * * T T * * L L 15 P 22 L 23 C 30 M 36 K 68 R 72 E 77 C 88 I 94 L 99 104 108 147 157 166 169 193 196 209 210 T N P Y T A L R R E S I S P Q Y R I T G R V V V V V V V V V V V V V S S A - - L L 105 H L C no odon A F121243 S ple am N 1-III N 1-III N 1-III N 1-III N 1-III N 1-III N 1-III C no lone 12/22 1/22 1/22 1/22 2/22 2/22 3/22 C no odon A F121243 S ple am N 1-V N 1-V N 1-V N 1-V N 1-V N 1-V N 1-V C no lone 9/21 1/21 1/21 2/21 2/21 3/21 3/21 P S H L 13 18 92 116 120 134 S Q G S V I H D V T Q V L G A H G L * * I H * K H F D M H L 33 35 46 50 116 120 129 164 182 D Y S S S V L P G A C V G C V G G L Q A * R L C L S T Q P * P * T G V I * F L L V * F L F I Q I K T T Y Q Q C C Q R A C 218 Codon no AF121243 Sample N3-I N3-I N3-I N3-I N3-I N3-I N3-I Codon no AF121243 Sample N2-I N2-I N2-I N2-I Codon no AF121243 Sample N2-II N2-II N2-II Codon no AF121243 Sample N3-II N3-II N3-II N3-II N3-II N3-II N3-II N3-II Clone no 12/20 6/20 1/20 1/20 V W Clone no 18/20 1/20 1/20 Codon no AF121243 Sample N2-III N2-III N2-III N2-III N2-III N2-III F L Clone no 12/20 3/20 2/20 1/20 1/20 1/20 Codon no AF121243 Sample N2-V N2-V N2-V N2-V 137 L 19 A Clone no 16/20 2/20 1/20 1/20 92 G 115 132 182 183 184 185 V F G R S P V S L V A L 10 I G P L 64 S T S P P * Clone no 11/22 1/22 1/22 1/22 1/22 1/22 1/22 1/22 1/22 2/22 1/22 Codon no AF121243 Sample N3-V N3-V N3-V N3-V N3-V N3-V N3-V N3-V T Clone no 13/20 1/20 1/20 1/20 1/20 1/20 1/20 1/20 Codon no AF121243 Sample N3-III N3-III N3-III N3-III N3-III N3-III N3-III N3-III N3-III N3-III N3-III 132 136 205 F C S Clone no 14/20 1/20 1/20 1/20 1/20 1/20 1/20 52 F Clone no 12/20 1/20 1/20 1/20 2/20 1/20 1/20 1/20 C d nn oo o 2 3 5 6 7 16 16 4 4 5 A114 F223 V S K L I P L S P S A E S S I S m le ap C n n lo e o N -II 80 /2 N -II 10 /2 A N -II 10 /2 F N -II 10 /2 E T N -II 10 /2 P P N -II 10 /2 T N -II 10 /2 P N -II 10 /2 S P P N -II 10 /2 V N -II 10 /2 N N -II 10 /2 N -II 10 /2 L N -II 10 /2 N C d nn oo o A114 F223 S m le ap C n n lo e o N -III /2 22 N -III 12 /2 N -III 12 /2 N -III 12 /2 N -III 12 /2 N -III 12 /2 N -III 12 /2 N -III 12 /2 N -III 12 /2 N -III 12 /2 N -III 12 /2 L T P H L K * L V S Y L S Y G 48 L K 8 17 16 16 18 19 23 L L D S A D S I T V 50 S S D 100 110 114 146 157 180 W S V E T R F R A G A A * G P Q 48 L 50 S 54 P 61 D L N S I 75 E 76 H * K S 111 143 179 189 190 209 R T R T P E R K I G G L A * L 54 P 70 A F L H S T H L * M C L G S D T L L S * D N I C L I 104 115 118 209 212 213 N V V E C P E D A A K Y L * W A T L 45 48 71 114 119 120 141 164 180 183 188 189 191 195 209 211 L L L V N V R P R R R T S R E Q Clone no 9/24 1/24 1/24 1/24 1/24 1/24 1/24 1/24 1/24 1/24 1/24 1/24 1/24 1/24 1/24 1/24 Codon no AF121243 Sam ple N5-II N5-II N5-II N5-II N5-II N5-II N5-II N5-II N5-II A * P 22 L 14 16 14 19 17 18 11 16 4 5 6 9 V E W P P I S R C T 26 W Codon no AF121243 Sam ple N5-I N5-I N5-I N5-I N5-I N5-I N5-I N5-I N5-I N5-I N5-I N5-I N5-I N5-I N5-I N5-I 110 141 151 173 209 212 S R F P E C L 46 S C d nn oo o 6 10 12 15 29 21 22 0 1 A114 F223 S A L R V F T E Q C S m le ap C n n lo e o N -I /2 10 N -I 20 /2 P A N -I 10 /2 T L A N -I 10 /2 P L Y N -I 20 /2 I N -I 30 /2 P 56 I Clone no 11/21 2/21 1/21 1/21 1/21 2/21 1/21 1/21 1/21 Codon no AF121243 Sam ple N5-III N5-III N5-III N5-III N5-III N5-III N5-III N5-III N5-III N5-III N5-III N5-III N5-III Clone no 7/21 2/21 1/21 1/21 2/21 1/21 1/21 1/21 1/21 1/21 1/21 1/21 1/21 Codon no AF121243 Sam ple N5-V N5-V N5-V N5-V N5-V N5-V N5-V N5-V N5-V N5-V N5-V N5-V Clone no 4/22 1/22 3/22 1/22 1/22 1/22 4/22 2/22 1/22 1/22 1/22 2/22 S P A D I Q G Q S S I P H Q H * C * * T L E Q * E G E L R E N N 46 47 59 81 89 106 144 159 181 191 211 213 S F L H L E V P R S Q T R G T L L S R Q * I I F T * P P T P P P P G C d nn oo o A114 F223 V C D L S m le ap C n n lo e o N -V 81 /2 N -V 11 /2 A N -V 11 /2 R N -V 11 /2 G N -V 11 /2 N -V 11 /2 N -V 11 /2 N -V 11 /2 N -V 11 /2 N -V 11 /2 N -V 11 /2 N -V 11 /2 N -V 11 /2 N -V 11 /2 C V G A M Y L * S L 5 8 9 11 18 19 10 17 12 12 28 29 20 21 5 0 1 0 1 S F S L R Q C M V P A S Y G R R E S Q 22 C P T S P K C P R Y W V L L S L * - - G K V * A W - - - - - - - - - R S * S K S Q H P M E D G N L L N M 14 16 25 29 48 51 61 62 99 153 154 164 184 204 205 207 210 212 C T G G L D D T T V W P S R S S S C R G A Q F E D R P G A I A A Q Q F Q * R H H S V P T V K I I I G M 11 35 47 57 73 75 93 115 145 146 159 164 165 183 187 209 212 S Y F R S E E V L E P P N R R E C E G P D Y P S G S A G S P Q Q Q G G Y * A V 219 Codon no AF121243 Sample R1-I R1-I R1-I R1-I R1-I R1-I R1-I R1-I R1-I R1-I R1-I R1-I R1-I R1-I R1-I R1-I R1-I H Clone no 12/28 1/28 1/28 1/28 1/28 1/28 1/28 1/28 1/28 1/28 1/28 1/28 1/28 1/28 1/28 1/28 1/28 Clone no 1/40 11/40 1/40 1/40 1/40 1/40 1/40 4/40 1/40 1/40 1/40 1/40 1/40 1/40 1/40 3/40 1/40 1/40 1/40 1/40 1/40 1/40 1/40 1/40 1/40 Codon no AF121243 Sample R1-IV R1-IV R1-IV R1-IV R1-IV R1-IV R1-IV R1-IV R1-IV R1-IV Clone no 11/20 1/20 1/20 1/20 1/20 1/21 1/20 1/20 1/20 1/20 33 D 34 P 48 L 51 D 67 Y 71 L 73 S 77 C 86 Q 88 I H 89 L 95 M 103 111 112 119 124 146 150 158 159 186 S R E N L E S P P R V S P T G G N G P G * * * * * S D D L 28 W T T T T T T * 29 G 34 P 46 S T * * L L L 48 L C F F H H H H I V V 50 S 51 D * L V V W W W V L L 57 R 67 Y 68 R H H H K A A V V V F C Y V V V V C C S 72 E 77 C 86 Q * D D V K K G Q Q 88 I 89 L 95 M D D D N G G * * * * * * * * * * * D D D D T T T T T T T T T T T L * P P N * * L V V L S S S L S S G E * * 107 108 112 115 116 126 131 141 146 149 151 154 159 179 185 186 D P E V S I W R E V F W P R P R A G G G L L L G G G G G G G L L L L L L L - - T T T 57 R T Q Q G * * A F F A F 50 S S S F F T H D Q Q W R P G G Y T T T M 70 A 72 E 80 H L * F L * S L * L L P S F - W H V V H H V V V V D D L S Y L S G V S F V G S V C S L G S I G G V R * V S F L I F S S A G S 65 A 72 E 79 P 83 A 87 A - S E D * E - I S R K S Q Q Q S S I S - S L G C I - - S S S - - R V S * H D V V D * C S R C D W W H * H H - L M Q L R G L T Q T S Q - - - * C Q S F Q * K * G H V V I V C G I * S I W E N G P - V W T W T * T L K * E * * E N K * S K 107 109 183 D A R I Y P Q Q G V Q R V K 18 Q Clone no 1/29 1/29 1/29 1/29 1/29 1/29 1/29 1/29 1/29 1/29 1/29 1/29 2/29 1/29 1/29 1/29 1/29 1/29 3/29 4/29 1/29 1/29 1/29 29 G 64 S Y 26 W Codon no AF121243 Sample R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III R1-II & III Codon no AF121243 Sample R1-V R1-V R1-V R1-V R1-V R1-V R1-V R1-V R1-V R1-V R1-V R1-V R1-V R1-V R1-V R1-V R1-V R1-V R1-V R1-V R1-V R1-V R1-V 28 W 20 S 28 W 37 E 38 F 45 L 47 F 51 D 55 S 61 D V T 101 103 112 115 116 119 126 128 136 139 162 164 167 168 178 179 180 184 185 V S E V S N I Q C F R P P I V R R S P M M M M Q Q Q Q Q Q Q Q Q Q G P A E A R N S G * * * * * * * I T K D S G S V P G L F G A - - G G G G - - L L L L - R - P P P P S - L T T T T T T Q Q Q Q Q Q - L 220 Codon no AF121243 S a m p le R -I R -I R -I R -I R -I R -I R -I R -I R -I R -I R -I R -I R -I R -I R -I R -I R -I C lo n e n o /3 /3 /3 /3 /3 /3 /3 /3 /3 /3 /3 /3 /3 /3 /3 /3 /3 L Codon no AF121243 S a m p le R -II & III R -II & III R -II & III R -II & III R -II & III R -II & III R -II & III R -II & III R -II & III R -II & III R -II & III R -II & III R -II & III R -II & III R -II & III C lo n e n o /2 2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 Codon no AF121243 S a m p le R -IV R -IV R -IV R -IV R -IV R -IV R -IV R -IV R -IV R -IV R -IV R -IV R -IV R -IV R -IV R -IV R -IV R -IV C lo n e n o /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 Codon no AF121243 S a m p le R -V R -V R -V R -V R -V R -V R -V R -V R -V R -V R -V R -V R -V R -V R -V R -V R -V C lo n e n o /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 23 C Codon no AF121243 Sample R3-II R3-II R3-II R3-II R3-II R3-II R3-II R3-II R3-II R3-II R3-II R3-II Clone no 1/21 3/21 2/21 1/21 1/21 1/21 1/21 1/21 1/21 1/21 3/21 5/21 Codon no AF121243 Sample R3-III R3-III R3-III R3-III R3-III R3-III R3-III R3-III R3-III R3-III R3-III R3-III Clone no 1/21 8/21 1/21 1/21 1/21 1/21 1/21 1/21 1/21 1/21 3/21 1/21 11 S Clone no 9/21 1/21 1/21 1/21 1/21 1/21 1/21 1/21 1/21 1/21 3/21 72 E 73 S 76 H 88 I 93 E 100 W 115 V 123 G 168 I 175 T 207 S Y G G F Q T G A T D A * C 25 G 28 W 31 D 34 P L I F S * G S A S L 66 L 42 V S F L 71 L 90 C 104 N 107 D 112 E 119 N 126 I 137 L 150 S 191 S E S * * * * * * * * * * * * T R 20 S 26 W N A A A A A A A A A A A A 47 F 48 L N 38 F C * * V L I F I S D D D D D D D D D D I F 64 S 69 E 76 H 77 C 81 H 92 G D G E S W 50 S L T R L L L L L L L L L L S Q 95 M 96 N P C Y L F W R A 103 S 116 S 126 I 128 Q 135 S 141 R 156 R 160 A 162 R Y P C C L P G Y S F T G P F G T G R 13 S 22 L C G 39 G 38 F * 47 F 56 I 59 L T P Q L S R L 21 K 30 M 43 E 44 L 49 P 52 F 56 I 61 D 72 E Y Y * * E E S S * * L L 75 E 85 R 90 C 92 G 96 N 115 V 116 S D D D D D D D D D D D D D D D D D P L L E A H H L L L D T T 118 V 121 N 134 I 137 L 149 V 163 P S Q Q 72 E 69 E V V V V V V V V V V V V V V V V V F F L L L 15 P * N T T R R A S A G F G G G G 164 P Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q I H G A F A S D T 167 P 173 P G T T T S S A G N N N Q F V V P T T T T T T T T T G H K K E 28 W 29 G 63 A 75 E 110 115 116 125 136 139 159 171 205 S V S K C F P T S R S N D N N N S K D T * 45 L * * * * * * * * * * * P 17 V 67 Y 89 L P H G 26 W A R 11 S 48 L 28 W Q * * * * * * * * * * * 51 D H 64 S 68 R N N N N N N N N N N N N * I A P K P 83 A R E T T T T T T T T T T T T G T G Y C C K 106 125 134 144 159 161 168 173 209 212 213 E K I V P Y I P E C W 56 I T T T T T T T 117 125 131 140 159 181 182 185 186 206 212 Y K W G P R G P R Q C H V 10 I 85 R N N N N N N N 85 R N N N N N N N N N N N N 93 E T A T T T T T T T T T T T T C D T R R S S 111 135 153 168 197 207 208 209 R S V I S S R E V T P P V T P W M Q F * Y S G Q G * Q F S Y V P I C onn od o 23 32 1 15 20 21 2 40 44 11 12 A 24 F 21 C I E G V S E S Q C S p amle C eno lon R 4-I 11 /2 R 4-I /20 R R 4-I /20 T R 4-I /20 Q R 4-I /20 E P R 4-I /20 A R 4-I /20 D L N V C onn od o 16 49 95 11 18 A 24 F 21 T P A C M E S S p amle C eno lon R 4-II 15 /2 R 4-II /20 I R 4-II /20 S V P R 4-II /20 G R 4-II /20 R 4-II /20 H * * T T T T T F V S K * G C onn od o 10 15 A 24 F 21 I P C S p amle C eno lon R 4-III /20 R 4-III /20 V R 4-III /20 Y R 4-III /20 R 4-III /20 R 4-III /20 R 4-III /20 R 4-III /20 R 4-III /20 S R 4-III /20 S R 4-III /20 S R 4-III /20 R 4-III /20 R 4-III /20 C onn od o A 24 F 21 S p amle C eno lon R 4-IV 13 /2 R 4-IV /20 R 4-IV /20 R 4-IV /20 R 4-IV /20 R 4-IV /20 R 4-IV /20 R 4-IV /20 L 18 08 T R A Q S 41 42 49 5 75 78 80 1 12 12 13 1 15 16 17 1 18 19 9 08 13 36 55 80 81 G S V P I L E S H Q P L I L F C I P P T R R S R V G P R R S S S S S Q Q G S S L L L P S S V V V I I I S Y T T T S S P Q * V F L C C C Q Q K L Q Q Q E F F F E H P A 16 18 19 2 36 11 12 L V I C P P S Q C S Q Q V T Y L S S S * W F C onn od o 42 54 A 24 F 21 V P I S p amle C eno lon R 4-V /20 S R 4-V /20 S L R 4-V /20 S R 4-V /20 V R 4-V 12 /2 V R 4-V /20 V V Q L L Y N V 11 15 16 08 97 13 L P L F P S L P Q P Q * * L L Q Q Q Q T K L K L 184 S P 122 125 150 159 179 205 209 211 212 M K S P R S E Q C L I Codon no AF121243 Sample R3-V R3-V R3-V R3-V R3-V R3-V R3-V R3-V R3-V R3-V R3-V 71 L E P 28 W Clone no 1/22 11/22 1/22 1/22 1/22 1/22 1/22 1/22 1/22 1/22 1/22 1/22 58 D I 26 W Codon no AF121243 Sample R3-IV R3-IV R3-IV R3-IV R3-IV R3-IV R3-IV R3-IV R3-IV R3-IV R3-IV R3-IV 51 D G 10 I L 41 S R 13 S Clone no 1/20 1/20 1/20 1/20 1/20 4/20 1/20 1/20 1/20 1/20 1/20 2/20 1/20 2/20 1/20 39 G P M Codon no AF121243 Sample R3-I R3-I R3-I R3-I R3-I R3-I R3-I R3-I R3-I R3-I R3-I R3-I R3-I R3-I R3-I 33 D S 221 C A S R R R R R R R R R R R R odon no F121243 a m p le -I -I -I -I -I -I -I -I -I -I -I -I 34 P C A S R R R R R R R R R R R R R odon no F121243 a m p le -II -II -II -II -II -II -II -II -II -II -II -II -II C lo n e n o /2 1 /2 /2 1 /2 1 /2 1 /2 1 /2 1 /2 /2 1 /2 1 /2 1 /2 /2 C A S R R R R R R R R R R R odon no F121243 a m p le -III -III -III -III -III -III -III -III -III -III -III C lo n e n o /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 C A S R R R R R R R R R R R odon no F121243 a m p le -IV -IV -IV -IV -IV -IV -IV -IV -IV -IV -IV C A S R R R R R R R R R R odon no F121243 a m p le -V -V -V -V -V -V -V -V -V -V C lo n e n o /2 /2 /2 /2 /2 /2 /2 /2 2 /2 /2 /2 2 /2 62 T 71 L 89 L 93 E V V G 115 V 164 P 187 R 209 E A A T T T T T T A I K C P * V V C 34 P 48 L 50 S 51 D Q Q S S Q E N 52 F 53 F 57 R 59 L 86 Q 89 L 105 L 111 R 113 L 122 M - - - - - - L L 28 W L 136 C 152 G 162 R - S P P * * K 19 A 19 A T T T T T T T T T T T 34 P 26 W V V V * P S W T T * 186 R 192 P K S A F * F L * L L S L S P S S A V S G F C G Q 51 D 79 P 80 H 103 S 117 Y 135 S 137 L 150 S 170 S 176 T 177 V P A R G 45 L E F T T T T Y Q 66 L S S S S S S S S S S S 73 S R P G H F 75 E T 79 P 84 L 89 L 123 G V P P P P P P P P P P P 125 K * S I D G Y L S L 183 R 204 R 212 C I G K 127 R D 132 F 141 R S 150 S S 174 E 175 T P P L L S S P T T T T G F 34 P 53 F 78 S P 89 L 90 C P V T V V V D 130 L 164 P 184 S Q P P 206 Q 207 S S P K R R 125 K M 24 L 206 Q I G T T 205 S P G G F C lo n e n o /2 /2 /2 /2 2 /2 2 /2 /2 /2 2 /2 /2 48 L H 27 L C lo n e n o /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 35 Y K K 190 P R 197 S P 211 Q K P H R F S L Y Y P P G G * * T T S S K K S S A A G G P I I Figure 32 Amino acid changes in deduced HBV precore amino acid sequences of the viral quasispecies Only aa changes are displayed Clones that had the same aa sequence as reference (NCBI Gene Bank, Accession No AF121243) are highlighted with light green, clones with frame shifts are highlighted with orange, stop codon mutations with pink, insertions with red and deletions are indicated as dashes highlighted with light blue Numbers of identical clones and total numbers of clones from each sample are shown 222 Appendix B: Reagents and Equipment Source of chemicals and reagents BDH LABORATORY SUPPLIES, Poole, Dorset, England: Glycerol, paraformaldehyde (PFA), Tris-HCl, Tris-base (Trizma) BECTON DICKINSON, Franklin Lakes, USA: BactoTM Agar BIO-RAD LABORATORIES, Hercules, USA: Acrylamide/bisacrylamide, agarose, ammonium peroxide sulphate, mixed bed resin, N,N,N,N-tetramethyl-ethylenediamine (TEMED) and urea BMA, ROCKLAND, USA: Gelstar nucleic acid gel stain CALBIOCHEM, Darmstadt, Germany: Mounting media DECKGLASER, Germany: Slides and coverslips E MERK, Germany: Absolute alcohol, hydrochloric acid, methanol FINNZYMES OY, Espoo, Finland: DYNAzyme EXTTM Taq DNA polymerase FMC BIOPRODUCTS, Rockland, USA: NuSieve agarose 223 GENSET Singapore Biotech Pet ltd, Singapore: All primers GIBCOBRL Life Technologies, Carlsbad, USA: kb plus DNA ladder, L-glutamine, MEM-non-essential amino acid, MEM-sodium pyruvate trypsin-EDTA NATIONAL UNIVERSITY MEDICAL INSTITUTES (NUMI), laboratory supply: autoclaved Dulbecco’s Modified Eagle’s Media (DMEM), 10 × TAE buffer (pH 8), × TBE buffer (pH 8.3), 1M Tris (pH 7.4), × PBS buffer (pH 7.5), 10 × PBS buffer (pH 7.5) and LB broth NEW ENGLAND BIOLABS, USA: 100 bp DNA ladder, 1kb DNA ladder, restriction enzymes with buffers and T4 DNA ligase with buffer PE BIOSYSTEMS, USA: Blue Dextran/EDTA (sequencing loading dye) PROMEGA, Madison, USA: dATP, dCTP, dGTP, dTTP SIGMA CHEMICAL COMPANY, USA: ampicillin, β-mercaptoethanol, bovine serum albumin, bromophenol blue, calcium chloride, Diethyl Pyrocarbonate (DEPC), ethidiumbromide, formamide, isopropylthio-β-D-galactoside (IPTG), isopropanol, sodium acetate, sodium chloride, sodium deodecyl sulphate (SDS), ethylene diamine tetra-acetic acid (EDTA), 5-bromo-4-chloro-3-indolyl- β-D-galactoside (X-gal), saponin, and xylene cyanol; Tritc conjugated rabbit anti-mouse Ig-G antibody 224 US BIOLOGICAL, USA: Mouse anti-hepatitis B e antigen Source of kits and systems ABBOTT LABORATORIES, USA: Anti-delta EIA diagnostic kit, anti-HBe EIA diagnostic kit, HBe 2.0 IMX diagnostic kit and HIV-EIA diagnostic kit BIO 101, USA: Geneclean II kit MILLIPORE, USA: Syringe filter (0.22 µm) INVITROGEN, USA: Superscript RT-PCR kit, pcDNA6/V5-His vector PE BIOSYSTEMS, USA: ABI Prism BigDye terminator cycle sequencing ready reaction kit PROMEGA, Madison, USA: Wizard Miniprep kit, pGEM-T and pGEM-T easy vector systems, β-galactosidase enzyme assay system with reporter lysis buffer ROCHE, Germany: Amplicor HBV monitorTM test kit QIAGEN, Germany: DNA Blood Mini kit, Effectene transfection kit, Hot star Taq PCR kit and RNeasy mini kit 225 Source of equipment ABBOTT LABORATORIES (USA): IMXSYSTEM B BRANUN BIOTECH INTERNATIONAL: Shaker incubator (CERTOMAT BS-1) BECTON DICKINSON, USA: Petri dish, cell culture plates and flasks, 15ml and 50 ml Falcon tubes, ml, 10ml and 25ml of pipettes, and pipet-aid with T.C filter Beckman, USA: AllegraTM 64 R Centrifuge BIO-RAD, USA: Power/PAC 200; Horizontal wide mini-sub cell GT, mini-sub cell GT, and the DcodeTM universal mutation detection system EPPENDORF, Hamburg, Germany: Centrifuge 5415C, centrifuge 5415D, Concentrator 5301, 1.5 ml microcentrifuge tubes and 0.2ml of PCR tubes GIBCO BRL, Carlsbad, USA: Electrophoresis power supply 250EX HOEFER PHARMACIA BIOTECH INC, USA: Hoefer SG100 Gradient maker HAMLTON, USA: Multiple channel syringes HIRAYAMA, Japan: Autoclave (HVE-50) 226 IMPROVED NEUBAUR, USA: Hausser Scientific hemocytometer KODAK, USA: Polarid camera; Polaroid T 667 film, film developer and x-ray film cassette NALGE COMPANY, USA: Bottle filter (0.22 µm) NESLAB endocal, USA: Water bath NUAIRTM , USA: Biological safety cabinet Class II (NU-440-400E) OLYMPUS, Japan: Olympus IX 70 inverted microscope and SLUOVIEW 300 confocal microscope PERKIN ELMER (USA): ABI PRISM 377 sequencer and ABI PRISM 377 XL collector sequencing analysis software version 3.41 PHARMACIA BIOTECH, England: Ultro spec 2000 spectrophotometer SANYO electric Co Ltd.: CO2 Incubator TECAN Group Ltd., Maennedorf, Switzerland: Sunrise - absorbance reader THERMOLYNE, USA: Vortex (Maxi MixII 37600 Mixer) 227 VIBER LOUR MAT, France: UV transilluminator WHATMAN BIOMETRA, Maidstone, UK: T3 Thermocycler (DNA cycler) 228 Appendix C: Buffers and solutions 10% (w/v) Ammonium peroxide sulphate (APS) g of ammonium peroxide dissolved in 10 ml of distilled water and stored in the dark at – 20oC until use Ampicillin stock solution Ampicillin was dissolved in distilled water to give a final concentration of 100 mg/ml The solution was then filtered with a 0.22 µm syringe filter and stored at – 20oC Complete Dulbecco’s Modified Eagle’s Media (DMEM) 10 ml of L-glutamine (100 ×), MEM-non-essential amino acid (100 ×), PenicillinStreptomycin (100 ×), MEM- sodium pyruvate solution (100 ×) and 100 ml of fetal bovine serum were added in the 860 ml DMEM supplied by NUMI and mixed well before use 10 x DNA Loading Buffer 10ml ml Glycerol, mg of bromophenol blue, 400 µL of 0.5 M EDTA were mixed with distilled water to a final volume of 10 ml and stored at room temperature Ethidium bromide stock solution (1%) 0.1 g Ethidium bromide was dissolved in 100 ml of distilled water and stored at 4oC in the dark 229 Gelstar stain solution for DGGE gel µl of Gel star was mixed well with 50 ml of × TAE buffer, pH before use IPTG stock solution (0.2M) 1.2 g IPTG dissolved in 25 ml of distilled water was filtered and stored at oC LB Agar 7.5 g of agar were dissolved in 500 ml of LB broth and autoclaved LB broth with ampicillin 500 µl of 100 mg/ml ampicillin solution were added into 500 ml of LB broth to give a concentration of 0.1 mg/ml It was prepared when required LB agar with ampicillin 500 µl of 100 mg/ml stock ampicillin solution were added to 500 ml of autoclaved LB agar liquid (around 50 oC) and mixed well before pouring the plates LB agar with ampicillin, X-gal and IPTG 1.25 ml of IPTG stock solution and 0.8 ml of X-Gal stock solution were added into the LB agar with ampicillin liquid and mixed well to pour the plates 230 0.1 M CaCl2 3.675g of CaCl2 dissolved in distilled water to a final volume of L was autoclaved and stored at oC until required 0.2 M EDTA, pH 8.0, ml ml of 0.2 M EDTA pH 8.0 and 400 µl of 0.5 M EDTA were mixed with 600 µl of double distilled water 3.7% PFA 10 ml 3.7 g of PFA was dissolved with 10 ml of × PBS pH 7.4 0.1% Saponin 0.1 g of saponin was dissolved in × PBS TE (Tris/EDTA) Buffer pH 7.4, 100 ml ml of M Tris, pH 7.4, (10mM) and 0.2 ml of 0.5M EDTA (pH 8.0) (1mM) were mixed and topped up to 100 ml with double distilled water X-Gal stock 100 mg of 5-bromo-4-chloro-3-indolyl-β-D-galactoside (x-gal) were dissolved in ml of N,N’- dimethylformamide and stored at -20oC in the dark 231 ... 22 88 Pro to Thr 29 Transition C to T 22 89 30 Transition C to T 22 90 31 Deletion 105 nt 21 50 -22 54 32 Deletion 130 nt 22 04 -23 33 33 Deletion 105 nt 21 34 -22 38 34 *aa change not indicated because the... of HBV DNA associated with HBeAg SC in chronic hepatitis B patients HBV mutations have been intensively studied in different types of hepatitis B patients in recent years because it has been believed... 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,