慢性B型肝炎是重要的健康問題，患者通常為肝硬化、肝臟代償失調以及肝癌的高危險群。目前已有數種核苷類似物用來治療慢性B型肝炎，但治療必需長期的服用，而抗藥性通常會在一到數年之後發生。先前的研究顯示病毒quasispecies演化和慢性病毒感染的致病機轉有著密切的關係，在發生e抗原陰轉的慢性B型肝炎患者體內，其病毒多樣性和未發生e抗原陰轉的患者有顯著的不同。然而目前對於長期核苷類似物治療的慢性B型肝炎患者體內病毒quasispecies演化仍不清楚，因此本研究將嘗試釐清在連續接受核苷類似物治療的患者體內，病毒的演化行為和產生抗藥性之間的關係。本研究收集連續服用干安能以及貝樂克後都產生抗藥性的病人作為實驗組，另外收集在連續用藥過程中只有產生干安能抗藥性的病人作為作為對照組，每位患者各別於六個時間點收集血清檢體：(I)服用干安能前 (II)服用干安能後六個月 (III)發生干安能抗藥性時 (IV)服用貝樂克前 (V)服用貝樂克後六個月 (VI)發生貝樂克抗藥性時。所有的檢體針對B型肝炎病毒反轉錄酶區域片段進行選殖以及定序，每個檢體挑選二十個選殖菌落進行定序，所得到的序列將用來進行病毒多樣性、演化樹譜以及抗藥性突變等分析。實驗結果顯示，會產生貝樂克抗藥性的患者在干安能抗藥性產生後，體內病毒多樣性有明顯的增加。會產生貝樂克抗藥性的患者在第四時間點時，體內病毒多樣性比在第三時間點時高出2.5倍(P=0.031)，另外也比貝樂克治療有反應的患者在相同時間點之病毒多樣性高出2倍(P=0.042)。病毒多樣性的顯著升高可能和患者對於貝樂克產生抗藥性有關，除此之外，這樣的現象或許能應用在預測貝樂克抗藥性的產生。

Chronic hepatitis B virus (HBV) infection is a worldwide health problem. Patients with chronic hepatitis B are at increased risk of developing cirrhosis, hepatic decompensation, and hepatocellular carcinoma (HCC). Several nucleoside analogues could be used as antiviral therapy, but the long-term therapy is necessary and the antiviral resistance develops one or several years later. Previous studies showed that long-term viral quasispecies evolutionary behavior is different between HBeAg seroconverters and nonseroconverters. At present, little is known about viral quasispecies evolution in the development of antiviral resistance after long-term treatment. This study is conducted to clarify the long-term viral evolutionary behavior in patients receiving sequential nucleoside analogues therapy. Chronic hepatitis B patients with lamivudine and entecavir resistance after receiving sequential lamivudine and entecavir therapy were enrolled as the study group, and then chronic hepatitis B patients receiving long-term entecavir therapy after development of lamivudine resistance were enrolled as the control group. Serum samples were collected for each patient at six time points: (I) before lamivudine treatment (II) six months after lamivudine treatment (III) occurrence of lamivudine resistance (IV) before entecavir treatment (V) six months after entecavir treatment (VI) occurrence of entecavir resistance. All of the samples were used for cloning and sequencing of the reverse transcriptase region. At least 20 colonies were selected and sequenced for each sample. All of the sequences are used for analysis of viral diversity, phylogenetic analysis and antiviral resistant mutation. The data showed that, after occurrence of lamivudine resistance, patients progressing entecavir resistance showed a striking increase in viral diversity. At the time point IV, patients progressing entecavir resistance had 2.5-fold higher viral diversity than at the time point III (P=0.031) and 2-fold higher viral diversity than patients responding to entecavir treatment at the same point (P=0.042). Viral diversity increased significantly may be associated with the coming of entecavir resistance and be used to predict the occurrence of entecavir resistance.

1.MacCallum FO. Homologous Serum Hepatitis. Lancet 1947;250:691-2.
2.Blumberg BS, Gerstley BJ, Hungerford DA, London WT, Sutnick AI. A serum antigen (Australia antigen) in Down's syndrome, leukemia, and hepatitis. Ann Intern Med 1967;66:924-31.
3.Prince AM. An antigen detected in the blood during the incubation period of serum hepatitis. Proc Natl Acad Sci U S A 1968;60:814-21.
4.Okochi K, Murakami S. Observations on Australia antigen in Japanese. Vox Sang 1968;15:374-85.
5.Dane DS, Cameron CH, Briggs M. Virus-like particles in serum of patients with Australia-antigen-associated hepatitis. Lancet 1970;1:695-8.
6.Almeida JD, Rubenstein D, Stott EJ. New antigen-antibody system in Australia-antigen-positive hepatitis. Lancet 1971;2:1225-7.
7.Magnius LO, Espmark JA. New specificities in Australia antigen positive sera distinct from the Le Bouvier determinants. J Immunol 1972;109:1017-21.
8.Okamoto H, Tsuda F, Sakugawa H, Sastrosoewignjo RI, Imai M, Miyakawa Y, Mayumi M. Typing hepatitis B virus by homology in nucleotide sequence: comparison of surface antigen subtypes. J Gen Virol 1988;69 ( Pt 10):2575-83.
9.Kramvis A, Kew M, Francois G. Hepatitis B virus genotypes. Vaccine 2005;23:2409-23.
10.Le Bouvier GL, McCollum RW, Hierholzer WJ, Jr., Irwin GR, Krugman S, Giles JP. Subtypes of Australia antigen and hepatitis-B virus. JAMA 1972;222:928-30.
11.Knipe D, Howley P. Hepadnaviridae and their replication. Fields' virology(5th editon) 2007.
12.Robinson WS, Lutwick LI. The virus of hepatitis, type B. N Engl J Med 1976;295:1168-75.
13.Okamoto H, Yotsumoto S, Akahane Y, Yamanaka T, Miyazaki Y, Sugai Y, Tsuda F, Tanaka T, Miyakawa Y, Mayumi M. Hepatitis B viruses with precore region defects prevail in persistently infected hosts along with seroconversion to the antibody against e antigen. J Virol 1990;64:1298-303.
14.Summers J, O'Connell A, Millman I. Genome of hepatitis B virus: restriction enzyme cleavage and structure of DNA extracted from Dane particles. Proc Natl Acad Sci U S A 1975;72:4597-601.
15.Rehermann B, Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection. Nat Rev Immunol 2005;5:215-29.
16.Will H, Reiser W, Weimer T, Pfaff E, Buscher M, Sprengel R, Cattaneo R, Schaller H. Replication strategy of human hepatitis B virus. J Virol 1987;61:904-11.
17.Tuttleman JS, Pourcel C, Summers J. Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell 1986;47:451-60.
18.Ganem D, Prince AM. Hepatitis B virus infection--natural history and clinical consequences. N Engl J Med 2004;350:1118-29.
19.McMahon BJ, Alward WL, Hall DB, Heyward WL, Bender TR, Francis DP, Maynard JE. Acute hepatitis B virus infection: relation of age to the clinical expression of disease and subsequent development of the carrier state. J Infect Dis 1985;151:599-603.
20.Fattovich G. Natural history and prognosis of hepatitis B. Semin Liver Dis 2003;23:47-58.
21.Chan HL, Leung NW, Hussain M, Wong ML, Lok AS. Hepatitis B e antigen-negative chronic hepatitis B in Hong Kong. Hepatology 2000;31:763-8.
22.Lok AS, McMahon BJ. Chronic hepatitis B. Hepatology 2007;45:507-39.
23.Lampertico P, Del Ninno E, Vigano M, Romeo R, Donato MF, Sablon E, Morabito A, Colombo M. Long-term suppression of hepatitis B e antigen-negative chronic hepatitis B by 24-month interferon therapy. Hepatology 2003;37:756-63.
24.Lau GK, Piratvisuth T, Luo KX, Marcellin P, Thongsawat S, Cooksley G, Gane E, Fried MW, Chow WC, Paik SW, Chang WY, Berg T, Flisiak R, McCloud P, Pluck N. Peginterferon Alfa-2a, lamivudine, and the combination for HBeAg-positive chronic hepatitis B. N Engl J Med 2005;352:2682-95.
25.Ghany M, Liang TJ. Drug targets and molecular mechanisms of drug resistance in chronic hepatitis B. Gastroenterology 2007;132:1574-85.
26.Lai CL, Chien RN, Leung NW, Chang TT, Guan R, Tai DI, Ng KY, Wu PC, Dent JC, Barber J, Stephenson SL, Gray DF. A one-year trial of lamivudine for chronic hepatitis B. Asia Hepatitis Lamivudine Study Group. N Engl J Med 1998;339:61-8.
27.Dienstag JL, Schiff ER, Wright TL, Perrillo RP, Hann HW, Goodman Z, Crowther L, Condreay LD, Woessner M, Rubin M, Brown NA. Lamivudine as initial treatment for chronic hepatitis B in the United States. N Engl J Med 1999;341:1256-63.
28.Ryu SH, Chung YH, Choi MH, Kim JA, Shin JW, Jang MK, Park NH, Lee HC, Lee YS, Suh DJ. Long-term additional lamivudine therapy enhances durability of lamivudine-induced HBeAg loss: a prospective study. J Hepatol 2003;39:614-9.
29.Fung SK, Chae HB, Fontana RJ, Conjeevaram H, Marrero J, Oberhelman K, Hussain M, Lok AS. Virologic response and resistance to adefovir in patients with chronic hepatitis B. J Hepatol 2006;44:283-90.
30.Chae HB, Hann HW. Time for an active antiviral therapy for hepatitis B: An update on the management of hepatitis B virus infection. Ther Clin Risk Manag 2007;3:605-12.
31.Chang TT, Gish RG, de Man R, Gadano A, Sollano J, Chao YC, Lok AS, Han KH, Goodman Z, Zhu J, Cross A, DeHertogh D, Wilber R, Colonno R, Apelian D. A comparison of entecavir and lamivudine for HBeAg-positive chronic hepatitis B. N Engl J Med 2006;354:1001-10.
32.Lai CL, Shouval D, Lok AS, Chang TT, Cheinquer H, Goodman Z, DeHertogh D, Wilber R, Zink RC, Cross A, Colonno R, Fernandes L. Entecavir versus lamivudine for patients with HBeAg-negative chronic hepatitis B. N Engl J Med 2006;354:1011-20.
33.Lai CL, Leung N, Teo EK, Tong M, Wong F, Hann HW, Han S, Poynard T, Myers M, Chao G, Lloyd D, Brown NA. A 1-year trial of telbivudine, lamivudine, and the combination in patients with hepatitis B e antigen-positive chronic hepatitis B. Gastroenterology 2005;129:528-36.
34.Lai CL, Gane E, Liaw YF, Hsu CW, Thongsawat S, Wang Y, Chen Y, Heathcote EJ, Rasenack J, Bzowej N, Naoumov NV, Di Bisceglie AM, Zeuzem S, Moon YM, Goodman Z, Chao G, Constance BF, Brown NA. Telbivudine versus lamivudine in patients with chronic hepatitis B. N Engl J Med 2007;357:2576-88.
35.Nowak MA, Bonhoeffer S, Hill AM, Boehme R, Thomas HC, McDade H. Viral dynamics in hepatitis B virus infection. Proc Natl Acad Sci U S A 1996;93:4398-402.
36.Allen MI, Deslauriers M, Andrews CW, Tipples GA, Walters KA, Tyrrell DL, Brown N, Condreay LD. Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Lamivudine Clinical Investigation Group. Hepatology 1998;27:1670-7.
37.Das K, Xiong X, Yang H, Westland CE, Gibbs CS, Sarafianos SG, Arnold E. Molecular modeling and biochemical characterization reveal the mechanism of hepatitis B virus polymerase resistance to lamivudine (3TC) and emtricitabine (FTC). J Virol 2001;75:4771-9.
38.Yang H, Westland CE, Delaney WEt, Heathcote EJ, Ho V, Fry J, Brosgart C, Gibbs CS, Miller MD, Xiong S. Resistance surveillance in chronic hepatitis B patients treated with adefovir dipivoxil for up to 60 weeks. Hepatology 2002;36:464-73.
39.Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, Chang TT, Kitis G, Rizzetto M, Marcellin P, Lim SG, Goodman Z, Ma J, Brosgart CL, Borroto-Esoda K, Arterburn S, Chuck SL. Long-term therapy with adefovir dipivoxil for HBeAg-negative chronic hepatitis B for up to 5 years. Gastroenterology 2006;131:1743-51.
40.Angus P, Vaughan R, Xiong S, Yang H, Delaney W, Gibbs C, Brosgart C, Colledge D, Edwards R, Ayres A, Bartholomeusz A, Locarnini S. Resistance to adefovir dipivoxil therapy associated with the selection of a novel mutation in the HBV polymerase. Gastroenterology 2003;125:292-7.
41.Colonno RJ, Rose R, Baldick CJ, Levine S, Pokornowski K, Yu CF, Walsh A, Fang J, Hsu M, Mazzucco C, Eggers B, Zhang S, Plym M, Klesczewski K, Tenney DJ. Entecavir resistance is rare in nucleoside naive patients with hepatitis B. Hepatology 2006;44:1656-65.
42.Tenney DJ, Levine SM, Rose RE, Walsh AW, Weinheimer SP, Discotto L, Plym M, Pokornowski K, Yu CF, Angus P, Ayres A, Bartholomeusz A, Sievert W, Thompson G, Warner N, Locarnini S, Colonno RJ. Clinical emergence of entecavir-resistant hepatitis B virus requires additional substitutions in virus already resistant to Lamivudine. Antimicrob Agents Chemother 2004;48:3498-507.
43.Domingo E, Martinez-Salas E, Sobrino F, de la Torre JC, Portela A, Ortin J, Lopez-Galindez C, Perez-Brena P, Villanueva N, Najera R, et al. The quasispecies (extremely heterogeneous) nature of viral RNA genome populations: biological relevance--a review. Gene 1985;40:1-8.
44.Ross HA, Rodrigo AG. Immune-mediated positive selection drives human immunodeficiency virus type 1 molecular variation and predicts disease duration. J Virol 2002;76:11715-20.
45.Frost SD, Wrin T, Smith DM, Kosakovsky Pond SL, Liu Y, Paxinos E, Chappey C, Galovich J, Beauchaine J, Petropoulos CJ, Little SJ, Richman DD. Neutralizing antibody responses drive the evolution of human immunodeficiency virus type 1 envelope during recent HIV infection. Proc Natl Acad Sci U S A 2005;102:18514-9.
46.Lim SG, Cheng Y, Guindon S, Seet BL, Lee LY, Hu P, Wasser S, Peter FJ, Tan T, Goode M, Rodrigo AG. Viral quasi-species evolution during hepatitis Be antigen seroconversion. Gastroenterology 2007;133:951-8.
47.Chayama K, Suzuki Y, Kobayashi M, Tsubota A, Hashimoto M, Miyano Y, Koike H, Koida I, Arase Y, Saitoh S, Murashima N, Ikeda K, Kumada H. Emergence and takeover of YMDD motif mutant hepatitis B virus during long-term lamivudine therapy and re-takeover by wild type after cessation of therapy. Hepatology 1998;27:1711-6.