7 200 ; 5-33 B Lamivudine B YDD motif, WHO Abstract Lamivudine-Resistance in Patients with Chronic Hepatitis B and/or Cirrhosis and Detection of utations in YDD otif of Hepatitis B Virus Genome Sung Ho Kang,.D., Young in Park,.D., Jong Young Choi,.D., Je Hyun Shin,.D., Tae Wook Park,.D., Si Hyun Bae,.D., Byung Hun Byun,.D., Byung in Ahn,.D., Chang Don Lee,.D., Sang Bok Cha,.D., Kyu Won Chung,.D., Hee Sik Sun,.D., Doo Ho Park,.D., Boo Sung Kim,.D. Department of Internal edicine, College of edicine, and WHO Collaborating Center f or Ref erence and Research on Viral Hepatitis, The Catholic University of Korea, Seoul, Korea B ackg round/ A im s : Lamivudine is an antiviral nucleoside analogue effective for the treatment of hepatitis B virus (HBV) infection via the inhibition of DNA polymerase activity. T he mutations, however, in YDD motif, such as YVDD and YIDD, have been found to interfere with the therapeutic efficacy of lamivudine. T his study was performed to identify the role of such mutant- type HBV among Korean hepatitis B patient s with chronic hepatitis or cirrhosis receiving lamivudine treatment. ethods : Serum samples were collected from four groups of patients; patients with breakthrough (group I, n =8); patient s who showed no response after the treatment (group II, n = 6); patients who showed good response (group III, n = 6); patient s with chronic hepatitis B without any treatment (group IV, n = 4). utations were detected by PCR- cloning and automated sequencing. Results : utations in YDD were found in only 4 (50%) in group I and were negative in group II. No mutations could be identified in the serum samples collected before treatment and from groups III and IV. YVDD mutation was found to be associated with two additional mutations, L- to- in 528th amino acid and L- to- V in 577th amino acid. Conclusions : Lamivudine resistance appeared in three different patterns: () breakthrough related to the mutations in YDD motif; (2) breakthrough not related to the YDD mutations; and (3) primary non- responder not related to the YDD mutations.(korean J Hepatol 200;7 :5-33) Key W ords : Lamivudine, Hapatitis/ Viral/ Chronic Hepatitis B, Lamivudine- resistant, YDD motif 200074; 20002; 200029 Abbreviations: AFP, alpha-fetoprotein; ALT, alanine aminotransferase; anti-hbc, antibody to hepatitis B core antigen; anti-hbe, antibody to HBeAg; anti-hbs, antibody to HBsAg ; AST, aspartate aminotransferase; HBeAg, hepatitis e antigen ; HBV, hepatitis B Virus ; IFN-, interferon- alfa; PCR, polymerase chain reaction; YIDD, tyrosineisoleucine- aspartate- aspartate; YDD, tyrosine- methionine- aspartate- aspartate; YVDD, tyrosine- valine- aspartateaspartate. Young in Park.D. Department of Internal edicine, Kangnam St. ary s Hospital, College of edicine, T he Catholic University of Korea, # 505 Banpo- dong, Seocho- ku, 37-040 Seoul, Korea Phone: 590-2388, 2384; Fax : 536-9559; E- mail: ympark @cmc.cuk.ac.kr Roche(). 5
The Korean Journal of Hepatology : Vol. 7. No.. 200 B. B - (recombinant inter - feron- alpha, IFN-),,. 2,3, B(Hepatitis B Virus, HBV) nucleoside analogue. lamivudine ((- )- - L- 2',3'- dideoxy-3'- thiacytidine, 3TC) human immunodeficiency virus ( HIV) (reverse transcriptase), HBVRNA- dependent DNA, 4-7 B. 8-6, lamivudine HBV, DNA YDD (tyrosinemethionine- aspartate- aspartate amino acid) motif 552 methioninisoleucine ('- to- I', YIDD mutant) valine ('- to- V ', YVDD mutant). 7-2 hepadna (polymerase)ydd motif (polymerization). 22 Blamivudine, lamivudine HBV. lamivudine B YDD motif DNA lamivudine.. 24, 9954 lamivudine (Glaxo-Welcome, Inc., England) 50 mg( 30 )6 B HBV. ; (, breakthrough) HBV DNA aminotransferase, HBV DNA aminotransferase (n=8); (II, non-responder) 4HBV DNA, aminotransferase (n=6); (III, responder) HBV DNA aminotransferase (n=6)., lamivudine B (IV, n=4). I, II 6, III (, 2). 20 6, 4 3 B, 2. 20 lamivudine,,,,,,, 6
Sung Ho Kang, et al. Lamivudine-Resistance and Detection of utations in YDD otif of HBV Genome T able. Clinical Characteristics of Patients with Chronic Hepatitis B or Liver Cirrhosis Case No. 2 3 4 5 6 7 8 9 0 2 3 4 5 6 7 8 9 20 2 22 23 24 Age Sex Clinical Diagnosis 43 35 40 6 50 47 44 29 46 39 33 64 39 57 47 45 35 40 55 43 44 22 30 33 F F F F Cirrhosis B Cirrhosis B Cirrhosis B* Cirrhosis B Cirrhosis B Cirrhosis B Cirrhosis B Cirrhosis B C Histology before Treatment CAH, F4 CPH, F2 CAH, F2 CAH, F3 CAH, F3 CAH, F2 CPH, F0 CAH, F2 not done CPH, F0 CAH, F4 CAH, F F4 one CAH, F4 one one one one one CAH, F3 CPH, F0 CAH, F3 CAH, F3 HBeAg/Ab Status -/ + -/ + -/ + -/ + -/ + Protocol LA LA LA LA LA LA LA LA LA LAIFN LA LAIFN LA LA LA LA LA LA LA LA No No No No Treatment Duration (months) *HCC was developed in this compensated cirrhosis patient. CPH: chronic persistent hepatitis, CAH: chronic active hepatitis, F0: no fibrosis, F: fibrosis in portal area, F2: fibrosis in periportal area, F3: septal fibrosis or bridging fibrosis, F4: cirrhosis. +LA: lamivudine 50 mg per day. IFN: recombinant interferon-alpha 2a (LG BioTech, Seoul, Korea) 3U subcutaneous administration three times per week for 6 months. 4 4 2 5 4 0 5 7 4 3 0 7 4 4 5 4 2 7 2 4 2 3 2 8 HBV DNA., (chronic persistent hepatitis, CPH) (chronic active hepatitis, CAH). 23 CPH (mild degree), bridging necrosis CAH (moderate), bridging necrosis CAH (severe), ; 0 = no fibrosis; = mild fibrosis (portal expansion); 2 = moderate fibrosis (portal- portal septa); 3 = severe fibrosis (bridging with distortion); 4 = cirrhosis. 24 2. ) HBsAg, anti- HBs, HBeAg, anti-hbe, anti- HBc AFP (Abbott Laboratories, Abbott Park, IL, USA). 7
7 200 T able 2. Comparison of Some Clinical Features among the Patients Depending on the Response to the Treatment with Lamivudine Number of cases ale / Female edian age (range) yrs. HBeAg-positive/ -negative Decompensated cirrhosis histology before treatment Inflammatory activity* mild moderate severe Fibrosis stage F0-2 F3-4 Group I Group II Group III Group IV 8 6/ 2 44 (33-6) 5/3 2 6 4 5 6 5/ 44 (29-64) 6/0 5 4-5 0 6 5/ 43 (35-57) F0: no fibrosis, F: fibrosis in portal area, F2: fibrosis in periportal area, F3: septal fibrosis or bridging fibrosis, F4: cirrhosis. 6/0 4 2-4 4/0 32 (22-44) 2/ 2 0 4 2 3-705, 4 2,000 rpm 0 DNA, 75%, 5. 4 µl DNA, (polymerase chain reaction, PCR). Fig ure. An example of polymerase chain reaction targeting DNA polymerase gene region including YDD motif (309 bp in length). Sample #2 in lane 3 is negative for PCR. 2) DNA DNA DNA. 20 µl (0% SDS, 0N NaOH) 40 µl, 5, (3 KOAc, ph 5.2) 20 µl 40. phenol: chloroform (24:) 0 µl, 4 2,000 rpm 0, microfuge tube. 3 NaOAc (ph 5.6) 8 µl00% 300 µl 3) HBV DNAS, C HBV DNA S C dual- target PCR ; S primers (sense, nucleotide position 633-652, 5' - accagcacggg accatgcaa- 3' ; antisense, nucleotide position 500-59, 5' - ggcccccgcccataggaatc- 3'; amplicon size 54 bp), C primers (sense, nucleotide position 689-709, 5' - accttgaggcaaacttcaaa-3'; antisense, nucleotide position 2058-2078, 5'- cagaatagcttgcctgagtgc- 3'; amplicon size 390 bp). PCR (20 µl volume), DNA 4 µl6.2 µl (2 µl0x PCR <50 µ KCl, 0 m 8
3. Lamivudine B YDD motif PCR, 0 3 copies/ ml of serum ( A ). PCR. 25 Fig ure 2. (A) T o show the sensitivity of polymerase chain reaction, a plasmid vector, puc9, containing a full- length HBV DNA insert was serially diluted with distilled w ater and amplified using a primer set for detecting precore/ core gene region. HBV DNA copies of 03/ ml or 0.00 pg/ 20 µl in serum were detectable. (B) An example of dual target polymerase chain reaction. Precore/ core gene region (upper band, 390 bp in length) and surface gene (lower band, 54 bp in length) were amplified simultaneously. Sample #6 in lane 7 is negative for polymerase chain reaction and sample #7 in lane 8 is a negative control (distilled water). T he 23 ladder marker (GIBCO BRL) was loaded in lane as a ladder- size marker. Tris- HCl, ph 8.3,.5 m gcl2, 0.00% gelatin>, 200 µdntp <datp, dctp, dgtp, dt T P>, sense antisense primers 50 p, 0.4 unittaq DNA (Amersham & Pharmacia Biotech UK, Ltd., Amersham Place, England). Thermocycler (odel 2400, Perkin- Elmer Co., Foster, CA. USA), 40 : (985 denaturation, 55 30 annealing, 72 extension). 727. PCR 5 µlx TAE (0.8 Tris, 0.4 NaOAc, and 0.04 Na2EDTA) 2% agarose gel (GIBCO BRL, Gaithersburg, D, USA) (50 ma, 00 volts, 30), ethidium bromide (0.5 µg/ml), UV transilluminator amplicon ( B). HBV DNA, HBV DNA 4) HBV DNA YDD motif YDD motif (nucleotide position 738-749) HBV DNA primer (sense, nucleotide position 593-64, 5'- acttgcacttgtattcc catc- 3' ; antisense, nucleotide position 873-894, 5' - ccaattacatatcccatgaag- 3' ; amplicon size 309 bp)(reference sequence: numbering from EcoR site). 26 PCR (50 µl volume), DNA 5 µl5 µl0x PCR (50 µ KCl, 0 m Tris- HCl, ph 8.3,.5 m gcl2, 0.00% gelatin), 200 µdntp, sense antisense primers 50 p, 0.5 unit Taq DNA (Amersham & Pharmacia Biotech UK LTD.), 30; (94 denaturation, 55 annealing, 72 2 extension). 727. 2% agrose gel, ethidium bromide, UV transilluminator amplicon( ). 5) PCR PCR, HBV DNA plasmid vector PCR 0 3 copies/ml (5 0-2 pg/ ml), PCR ( 2, A) ; < 0.00 pg/ 20 µl (< 0 3 copies/ml), - ; 0.00pg/ 20 µl ( 0 3 copies/ml), trace; 0.00.pg/ 20 µl ( 0 4 0 5 copies/ml), +;.0 pg/ 20 µl ( 0 6 copies/ml) 9
The Korean Journal of Hepatology : Vol. 7. No.. 200 T able 3. Changes in Serum Alanine Aminotransferase (ALT ) Value, HBeAg Titer, and HBV DNA Amount before and during Lamivudine Treatment Change in ALT level (IU/dL) Change in HBeAg titer Change in the amount (pg) of HBV DNA in 20 L of serum File No. Before treatment (maximum) During treatment (minimum) Before treatment (maximum) During treatment (minimum) Before treatment During treatment Group I 307 623 788 968 005 54 584 866 07 48 70 222 9 09 55 7 3 4 36 26 4 70 2 28 00.2 67.4.4 0.6 84.7 36.9 24.6 8.8 26.6.0 0.00..0 0.00. 0.00. 0.00. 0.00..0 Group II 78 28 46 652 76 999 75 62 209 3 85 46 35 23 73 80 39 24 7.2 58.6 85.9 07 03.7 33.9 07.9 86.5 3.9 6 47.7 03.0.0 0.00..0 0.00.0 0.00..0 0.00. 0.00..0 0.00 Group III 30 389 904 969 734 74 26 59 9 4 03 82 39 2 4 26 24 30 4.2 06.3 23.6 25.6 6.0 64.9 7.2 2.3 9.6 4.9 2.4 3.2 0.00..0 0.00 000..0 0.00. : positive, : negative ++; 0 pg/ 20 µl ( 0 7 copies/ml) +++; 50 pg/ 20 µl (2.5 0 7 copies/ml) ++++. 6) PCR µlpge- T (Promega, adison, WI, USA), E. coli transformation - Gal/ IPTG, insert white colony. E. coli minipreparation (Wizard plus SV minipreps; Promega) plasmid DNA, DNA spectrophotometer 2-4 µg. Fluorescein- 5- isothiocyanate (FIT C)5`- T 7 SP6 primer dye termination, automated laser fluorescent sequencer (ALF, Amersham & Pharmacia Biotech UK, Ltd.). ( ) (DNASISWIN VERSION 2.0; Hitachi, Japan ).. ( )(n=6), 2(n=8), 3(n=6), (n=4) 43 20
Sung Ho Kang, et al. Lamivudine-Resistance and Detection of utations in YDD otif of HBV Genome (35-57), 44(33-6), 44(29-64), 32 (22-44), 4, 2,, 0. HBeAg 2 3 2. HBV DNA., 2, 3, 2, 6, 5, 4. 2. HBV DNA III (responder)pcr HBV DNA (0 3 copies/ ml 5 0-2 pg/ ml ), I, breakthrough, II HBV DNA (3). 4. YDD motif I (breakthrough) 8 YDD motif 552 () 4(50%), 4 (4). 4 2 YVDD YIDD(623 968 ), YVDD (788 ), YIDD (005 )(4). II (non- responder) 6, 6 HBV DNA YDD motif (4)., (n=20) (n=4)hbv DNA YDD (3,4 ; 3). 3. Breakthrough :,, I (breakthrough), lamivudine 4 (; 7-5 )breakthrough, HBeAg 5 HBV DNAHBeAg - 2. 3 lamivudine HBV DNA, HBeAg, 2 HBV DNA HBeAg., ( flare- up) 3(37.5%)(3). 6 5(83%) F3 F4., primary non-responder 6 5 F2, (2). 5. YDD Breakthrough lamivudine HBV I (breakthrough) YDD. 788, breakthrough 5 colony YVDD, lamivudine 2 colony (YDD), 8 colony, ( 4). 005, breakthrough 6 colony YIDD,, ( 4). 6. YDD YDD DNA 2
7 200 T able 4. Follow- up Duration, Serum Alanine Aminotransferase Level, HBeAg titer and Flare- up of Hepatitis at the Time of Breakthrough or Withdrawal, and Changes in YDD otif Sequence after Lamivudine Treatment in Group I and Group II File No. Group I Time breakthrough or withdrawal Flare-up of hepatitis ALT* HBeAg Change in YDD motif sequence after treatment 307 4 mo. no 54 36.3 3 YDD (of 3 colonies) 623 4 mo. yes 848 22.7 4 YVDD, 3 YIDD, and 3 YDD (of 0 colonies) 788 5 mo. yes 760.3 5 YVDD (of 5 colonies at breakthrough) / YVDD (of 2 colonies mo. after breakthrough) / 8 YDD (of 8 colonies 2 mo. after breakthrough) 968 5 mo. no 67 44.5 2 YVDD 2 YIDD (of 4 colonies) 005 4 mo. no 6 42 6 YIDD (of 6 colonies at breakthrough) / 2 YIDD (of 2 colonies 2 mo. after breakthrough) 54 7 mo. no 9 23.3 3 YDD (of 3 colonies) 584 0 mo. no 95 YDD (of 3 colonies) 866 8 mo. no 243 YDD (of 3 colonies) Group II 78 6 mo. no (until 2 mo.) 28 0 mo. no (until 0 mo.) 46 5 mo. no (until 8 mo.) 652 5 mo. yes (at 7 mo.) 76 4 mo. no (until 4 mo.) 999 7 mo. no (until 7mo.) 80 45.6 3 YDD (3 colonies) 23 9.9 3 YDD (3 colonies) 27 83.9 3 YDD (3 colonies) 36 65. 3 YDD (3 colonies) 40 84. 3 YDD (3 colonies) 38 23.5 3 YDD (3 colonies) *Peak alanine aminotrasferase level (IU/dL). All cases were positive for HBV DNA in serum by PCR before lamivudine treatment and at the time of withdrawal. All cases had wild type sequence in YDD motif sequence before lamivudine treatment. YVDD HBV DNA528 (L-to-) ( 3 623, 788 968 ), 2 577 (L- to- V) ( 3 623 968 )., YIDD 3(623, 788 005 ), 005 526 (TTT - to- TTC)546(T GT -to- GGT ) colony, 546(C- to- G) ( 3). HBV DNA pregenome encapsidation, RNA dependent- DNA, DNA- dependent DNA, RNase- H 3 HBV, 22
3. Lamivudine B YDD motif Fig ure 3. Sequence alignments of DNA polymerase gene region including YDD motif from serum HBV DNA isolated before lamivudine treatment and at the time of breakthrough or withdrawal of lamivudine because of primary non- response.( Continued next pag e) 23
The Korean Journal of Hepatology : Vol. 7. No.. 200 ( Continued next page ) 24
Sung Ho Kang, et al. Lamivudine-Resistance and Detection of utations in YDD otif of HBV Genome ( Continued next page ) 25
7 200 ( Continued next page ) 26
3. Lamivudine B YDD motif 27
The Korean Journal of Hepatology : Vol. 7. No.. 200 28
Sung Ho Kang, et al. Lamivudine-Resistance and Detection of utations in YDD otif of HBV Genome 29
7 200 catalytic domainydd motifhepadnavirus 22,27. Hepadnavirus, HBV, HBV DNA, DNA DNA covalently closed circular DNA (cccdna).. cccdna plusstrand pregenomic RNA Core DNA. minus strand DNA, RNase H pregenomic RNA, DNA- dependent DNA plus- strand DNA, nucleocapsid partially double- strand DNA. 28 triphosphatenucleocapsidhbv DNA DNA DNAchain terminator, duck hepatitis B virus Lamivudine 3'-TC-5'- lamivudinernadependent DNA DNA-dependent DNA chain terminator. 29 Lai 30 358 B lamivudine 00 mg52 HBeAg 56% B, Honkoop 5 3 B00 mg 300 mg HBV DNA., Grellier 4 B HBV lamivudine, Tassopoulos 3 HBeAg precore B 60 lamivudine 63%. lamivudinehbeag (precore ) (precore ) B HBV. Lai 30 4% lamivudine HBV, HBV DNA., HBeAg B27% 3, B 36% 32, 00% 2 0 lamivudine HBV. Woodchuck hepatitis virus ( WHV), lamivudine WHV, 9-2 lamivudine WHV. 33 lamivudine,. Lamivudine HBV DNA YDD motif, YIDDYVDD. HBVlamivudine, YDD HBV lamivudine 3-4 HBV. 34, YVDD 2, YIDD 2,. YVDD 528 L-to-, YIDD 30
3. Lamivudine B YDD motif, lamivudine. 35, YIDD YVDD HBV528L- to-. lamivudine YDD motif50%, YDD motif. breakthroughyvdd YIDD., breakthrough, YDD motif lamivudine., lamivudine, HBeAg, HBV DNA, YDD DNA, lamivudine HBV. breakthrough 4 (7-5 ), Lai 30 7-2Chayama 34 9-4, lamivudine breakthrough,. 3 breakthrough, F3- F4, 6 4,. Breakthrough, Lai 30., 2,. YVDDYIDD 4 2, Chayama 34 20., HBeAg precore HBV HBsAg B lamivudineydd HBeAg, 3 3 breakthrough. lamivudine YDD motif breakthrough, YDD motif breakthrough, YDD motif non - responder. : nucleoside analogue DNA HBV lamivudine B., 4-36% lamivudine 3
The Korean Journal of Hepatology : Vol. 7. No.. 200 HBV. YDD motifyvdd YIDD lamivudine. lamivudine B HBV DNA YDD motif. : lamivudine 50 mg6b, (HBV DNA, aminotransferase )(I, breakthrough), (II, non- responder), (III, responder) 8, 6, 6, B 4. HBV DNA, YDD motif,. : I YDD motif 552 () 4(50%), 4. 4 2 YVDDYIDD, YVDD, YIDD. II YDD motif. HBV DNA YDD. YVDD HBV DNA 528 (L-to-). : Lamivudine HBV genomeydd, (breakthrough) YDD, (non - responder)ydd. : / / B,, YDD motif. Hoofnagle JH, Shafritz DA, Popper H. Chronic type B hepatitis and the healthy HBsAg carrier state. Hepatology 987;7:758-763. 2. Honkoop P, de an RA, Niesters HG, et al. Quantitative hepatitis B virus DNA assessment by the limiting-dilution polymerase chain reaction in chronic hepatitis B patients: evidence of continuing viral suppression with longer duration and higher dose of lamivudine therapy. J Viral Hepat 998; 5:307-32. 3. Carithers RL. Effect of interferon on hepatitis B. Lancet 998;35:57. 4. Doong SL, Tsai CH, Schinazi RF, Liotta DC, Cheng YC. Inhibition of the replication of hepatitis B virus in vitro by 2',3' -dideoxy- 3'-thiacytidine and related analogues. Proc Natl Acad Sci USA 99;88:8495-8489. 5. Chang CN, Doong SL, Zhou JH, et al. Deoxycytidine deaminase- resistant stereoisomer is the active form of ( )- 2',3'-dideoxy-3'-thiacytidine in the inhibition of hepatitis B virus replication. J Biol Chem 992;267:3938-3942. 6. Chang CN, Skalski V, Zhou JH, Cheng YC. Biochemical pharmacology of (+)- and (- )- 2',3' -dideoxy-3'-thiacytidine as anti-hepatitis B virus agents. J Biol Chem 992;267: 2244-22420. 7. Charvet AS, Turin F, Faury P, et al. Synthesis and antiviral activity of new carbonylphosphonate 2',3'-dideoxy-3' -thiacytidine conjugates. Antiviral Res 994;25:6-68. 8. Benhamou Y, Dohin E, Lunel-Fabiani F, et al. Efficacy of lamivudine on replication of hepatitis B virus in HIVinfected patients. Lancet 995;345:396-397. 9. Dienstag JL, Perrillo RP, Schiff ER, Bartholomew, Vicary C, Rubin A. Preliminary trial of lamivudine for chronic hepatitis B infection. N Engl J ed 995; 333:657-66. 0. Bain VG, Kneteman N, a, et al. Efficacy of lamivudine in chronic hepatitis B patients with active viral replication and decompensated cirrhosis undergoing liver transplantation. Transplantation 996;62:456-462.. Benhamou Y, Katlama C, Lunel F, et al. Effects of lamivudine on replication of hepatitis B virus in HIVinfected men. Ann Intern ed 996;25:705-72. 2. Grellier L, utimer D, Ahmed, et al. Lamivudine prophylaxis against reinfection in liver transplantation for hepatitis B cirrhosis. Lancet 996;348:22-25. 3. Ben-Ari Z, Shmueli D, or E, Shapira Z, Tur-Kaspa R. Beneficial effect of lamivudine in recurrent hepatitis B after liver transplantation. Transplantation 997;63:393-396. 32
Sung Ho Kang, et al. Lamivudine-Resistance and Detection of utations in YDD otif of HBV Genome 4. Grellier L, Dusheiko G. Hepatitis B virus and liver transplantation: concepts in antiviral prophylaxis. J Viral Hepat 997; 4(Suppl ):- 6. 5. Honkoop P, de an RA, Zondervan PE, Schalm SW. Histological improvement in patients with chronic hepatitis B virus infection treated with lamivudine. Liver 997;7:03-06. 6. Lai CL, Ching CK, Tung AK, et al. Lamivudine is effective in suppressing hepatitis B virus DNA in Chinese hepatitis B surface antigen carriers: a placebo- controlled trial. Hepatology 997;25:24-244. 7. Balzarini J, Wedgwood O, Kruining J, et al. Anti-HIV and anti- HBV activity and resistance profile of 2',3'- dideoxy- 3'-thiacytidine(3TC) and its arylphosphoramidate derivative CF 09. Biochem Biophys Res Commun 996;225:363-369. 8. Ling R, utimer D, Ahmed, et al. Selection of mutations in the hepatitis B virus polymerase during therapy of transplant recipients with lamivudine. Hepatology 996;24:7-73. 9. Tipples GA, a., Fischer KP, Bain VG, Kneteman N, Tyrrell DL. utation in HBV RNA-dependent DNA polymerase confers resistance to lamivudine in vivo. Hepatology 996;24:74-77. 20. Bartholomew, Jansen RW, Jeffers LJ, et al. Hepatitis-B -virus resistance to lamivudine given for recurrent infection after orthotopic liver transplantation. Lancet 997;349:20-22. 2. Schalm SW. Clinical implications of lamivudine resistance by HBV. Lancet 997;349:3-4. 22. Toh H, Hayashida H, iyata T. Sequence homology between retroviral reverse transcriptase and putative polymerases of hepatitis B virus and cauliflower mosaic virus. Nature 983;305:827-829. 23. Liaw YF, Chu C, Chen TJ, Lin DY, Chang-Chien CS, Wu CS. Chronic lobular hepatitis: a clinicopathological and prognostic study. Hepatology 982;2:258-262 24. Desmet VJ, Gerber, Hoofnagle JH, anns, Scheuer PJ. Classification of chronic hepatitis: diagnosis, grading and staging. Hepatology 994;9:53-520 25. Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 989;339:237-238. 26. Okamoto H, T suda F, Sakugawa H, et al. Typing hepatitis B virus by homology in nucleotide sequence: comparison of surface antigen subtypes. J Gen Virol 988;69:2575-2583. 27. Radziwill G, Tucker W, Schaller H. utational analysis of the hepatitis B virus P gene product: domain structure and RNase H activity. J Virol 990;64:63-620. 28. Nassal, Schaller H. Hepatitis B virus replication. Trends icrobiol 993;:22-228. 29. Severini A, Liu XY, Wilson JS, Tyrrell DL. echanism of inhibition of duck hepatitis B virus polymerase by (- )-beta-l- 2',3' -dideoxy-3'-thiacytidine. Antimicrob Agents Chemother 995;39:430-435. 30. Lai CL, Chien RN, Leung NW, et al. A one-year trial of lamivudine for chronic hepatitis B. Asia Hepatitis Lamivudine Study Group. N Engl J ed 998;339:6-68. 3. Tassopoulos NC, Volpes R, Pastore G, et al. Efficacy of lamivudine in patients with hepatitis B e antigen- Negative/ Hepatitis B virus DNA- positive (Precore mutant) chronic hepatitis B. Hepatology 999;29:889-896. 32. Honkoop P, Niesters HG, de an RA, Osterhaus AD, Schalm SW. Lamivudine resistance in immunocompetent chronic hepatitis B. Incidence and patterns. J Hepatol 997;26: 393-385. 33. ason WS, Cullen J, oraleda G, et al. Lamivudine therapy of WHV-infected woodchucks. Virology 998;245:8-32. 34. Chayama K, Suzuki Y, Kobayashi, et al. Emergence and takeover of YDD motif mutant hepatitis B virus during long-term lamivudine therapy and re-takeover by wild type after cessation of therapy. Hepatology 998;27:7-76. 35. Allen I, Deslauriers, Andrews CW, et al. Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Lamivudine Clinical Investigation Group. Hepatology 998;27:670-677. 33