摘要

B型肝炎病毒（HBV）擁有放鬆、環形且不完整雙股，大約為3200個核苷酸的DNA基因體。目前可基於整個基因體序列差異到達8%以上的原則，而分為基因型A~H八型。在過去的研究中發現，大部分的基因型有其特定的地理分布，病毒的基因型也會影響慢性B型肝炎疾病的轉變。目前HBV基因分型的方法，主要有限制酶切割片段長度多樣性分析（RFLP）、基因型專一性引子法及直接定序法。但大多耗時、需要PCR後續的處理，甚或在我們資料庫預測上擁有很高的錯誤率。為了提昇基因分型方法的準確性，我們建立了由NCBI擷取的370株全長序列的HBV資料庫，並使用自行設計的資料庫連結軟體及Excel的公式作運算，去找出可以清楚分離HBV基因型A~G的合適設計點。我們設計了： (1)雜合式引子/探針組，藉由單一上機步驟的即時PCR及熔解曲線分析，同時完成病毒定量及基因型分組（ACDG vs. BEF）。在第二輪的即時PCR及熔解曲線分析中，再利用後續的引子/探針組，分別在ACDG及BEF群組中，鑑定出其所屬基因型；我們也設計了 (2)基因型專一性引子複合式PCR組（N3/ACDG），來輔助ACDG群組後續的分型鑑定（替代ACDG後續分型的探針）。在另一方面也設計了 (3)一套獨立的基因型專一性引子複合式PCR法（N1/ABFG及N2/CDE組），用以直接鑑定HBV A~G七個基因型。在110個臨床檢體（表面抗原及HBV DNA皆為陽性）測試的結果中，我們以自己設計的三套方法和前人發表的1998-RFLP法及2001-基因型專一性引子法作比較。其中2001-基因型專一性引子法只作20個檢體，原因是在先前實驗室的研究中，發現此法對於基因型B及C的鑑別力不佳。對於各方法間有爭議的檢體，則以直接定序法及加入HBV A~H八型參考基因型序列的樹狀圖分型法，來決定其所屬的基因型。各方法比較後顯示: (1)在熔解曲線分析法中，第一輪的即時PCR反應能夠定量病毒，並在103~1013 copies/mL病毒量範圍中擁有很好的回歸線性，同時能以不同的Tm值來區分基因型為ACDG及BEF兩群。第二輪的即時PCR暨熔解曲線分析則能將ACDG或BEF群作後續完整的分型，但在ACDG群組後續的分型鑑定上，則出現較多的錯誤。因為部分檢體在探針作用區序列上的變異，使得此法的準確度只達到80.9%。 (2)用來替代ACDG後續分型探針的N3/ACDG基因型專一性引子組，在ACDG群組後續的分型鑑定上明顯有效許多，並可將準確度提高至90.0%。 (3)獨立的基因型專一性引子複合式PCR法（N1/ABFG及N2/CDE組）也能成功並正確地鑑定出大部分檢體的基因型，準確率為83.6%，但其敏感度略遜於熔解曲線分析法。如果在扣除103~105 copies/mL極低病毒量的檢體後，此法甚至能達到98.5%的準確度。 (4)在前人發表方法中，1998-RFLP及2001-專一性引子法的準確率分別可達到88.2%及85.0%。（2001-專一性引子法中，若將總試驗檢體數提高至70個，其準確率則只有60.0%。）簡而言之，我們成功地設立了有效的HBV基因型分型方法，包含了即時PCR熔解曲線分析法及基因型專一性引子複合式PCR法。此外在熔解曲線分析法中，ACDG/BEF探針組除了能作為病毒定量的工具外，在應用於基因型B及C（亞洲地區主要的基因型）的初步預測上，亦具有很高的準確率（94.5%）。若有必要的話，可以再利用N1/ABFG及N2/CDE基因型專一性引子複合式PCR法，來進一步精準地鑑定出A~G七型的HBV基因型。我們在此提出的HBV基因分型實驗方法及設計，具有高偵測敏感度、同時能定量病毒及高準確率的特性，非常適合應用於臨床診斷或是HBV基因型相關的研究上。

Abstract

Hepatitis B virus (HBV) has a relaxed-circular, partially double stranded DNA genome of approximately 3200 nucleotides and has been classified into eight genotypes (A–H) based on an inter-group divergence of more than 8% in the entire genomic sequence. It has been shown that most HBV genotypes have distinct geographical distributions and that the viral genotypes may influence the clinical outcomes of chronic HBV infection. Currently, HBV genotyping is performed mainly by restriction fragment length polymorphism (RFLP) assay, genotype-specific primers assay, and direct sequencing. However, most of these methods are time-consuming or require post-PCR manipulations, and some even have high inaccuracy based on our database prediction. In order to improve the accuracy of genotyping assays, we analyzed 370 HBV full-length genomes containing all genotypes from NCBI, and used database-associated software designed by ourselves and Excel formulae to search for appropriate target sites for differentiation of genotypes A–G clearly. We designed (1) a hybridization primer/probe set for simultaneous HBV DNA quantification and differentiation of HBV genotypes into two groups (ACDG vs. BEF) by a single round real-time PCR procedure associated with melting curve analysis. Individual genotype in either ACDG- or BEF-groups can be further distinguished by another primer/probe set in a second round of real-time PCR reaction; we also designed (2) an improved multiplex PCR assay with genotype-specific primers (N3/ACDG set) to assist further differentiation of ACDG group instead of original ACDG-differentiated probe set. On the other hand, we designed (3) an independent method of multiplex PCR with genotype-specific primers assay (N1/ABFG and N2/CDE sets) to differentiate genotype A~G directly. To compare the assay accuracy of our established methods with RFLP and genotype-specific primers methods published in 1998 and 2001, 110 clinical samples which showed positive of HBsAg(+) and HBV DNA(+) were examined. Among all methods, only 20 samples were tested by the published genotype-specific primers method due to its poor accuracy in differentiating genotype B and C in our previous examination. Any discordant genotypes in dispute were verified by direct sequencing and phylogenetic tree assay with HBV A~H reference sequences. The comparisons showed that: (1) In melting curve analysis, the first round real-time PCR could quantify HBV DNA in a well linear regression range from 103 to 1013 copies/mL and separate genotypes ACDG from BEF with different melting temperatures. Then the second round of real-time PCR could identify the individual genotype in either ACDG- or BEF-group, but there were more errors occurred in the former group. The prediction of genotypes could reach 80.9% accuracy due to sequence variances within probe targeting sites. (2) The genotype-specific primers set (N3/ACDG) used in multiplex PCR instead of ACDG-separating probe used in the second round real-time PCR were effective in distinguishing individual genotype in the ACDG group and rescued the accuracy to 90.0%. (3) The genotype-specific primers (N1/ABFG & N2/CDE) used in multiplex PCR succeeded in differentiating most samples with 83.6% accuracy, despite having lower detection sensitivity than the melting curve protocol. Consequently, after excluding samples with very low viral loads (103~105 copies/mL), the accuracy of this independent protocol could reach 98.5% accuracy. (4) The previous RFLP (1998) and genotype-specific primers (2001) assays could reach 88.2% and 85.0% accuracy, respectively. (The latter method would have only 60.0% accuracy if examined samples number was expanded to 70.) In summary, we successfully established effective genotyping methods for HBV by real-time PCR with melting curve analysis and multiplex PCR with genotype-specific primers assay. In addition, the ACDG/BEF probe set can be applied to HBV DNA quantification and differentiation between HBV genotype B and C, the major genotypes in Asia, with high accuracy (94.5%) of the probe set in predicting these two genotypes. If necessary, the N1/ABFG plus N2/CDE genotype-specific primers set can be further used in distinguish all A~G genotypes with fidelity. The experimental protocol and methological designs reported herein will be extensively favorable for application in clinical diagnosis or genotype related researches with high sensitivity of detection, simultaneous HBV DNA quantification, and improved accuracy.

參 考 文 獻 (References)

Alter MJ. Epidemiology and Prevention of Hepatitis B. Semin Liver Dis 2003; 23:39-46.

Anderson TP, Werno AM, Beynon KA, and Murdoch DR. Failure To Genotype Herpes Simplex Virus by Real-Time PCR Assay and Melting Curve Analysis Due to Sequence Variation within Probe Binding Sites. J Clin Mircol 2003; 41:2135–2137.

Arauz-Ruiz P, Norder H, Robertson BH, and Magnius LO. Genotype H: a new Amerindian genotype of hepatitis B virus revealed in Central America. J Gen Virol 2002; 83:2059–2073.

Bartholomeusz A, and Schaefer S. Hepatitis B virus genotypes: comparison of genotyping methods. Rev Med Virol 2004; 14:3–16.

Berk PD, and Lok A. Hepatitis B: Progress in the Last Decade. Semin Liver Dis 2003; 23:1-4.

Block T M, Mehtal AS, Fimmel CJ, and Jordan R. Molecular viral oncology of hepatocellular carcinoma. Oncogene 2003; 22:5093–5107.

Blumberg BS, and Alter HJ. A “new” antigen in leukemia sera. JAMA 1965; 191:101–106.

Blut A. Hepatitis B Virus. Infus Ther Transfus Med 2000; 27:226-234.

Broderick AL, and Jonas MM. Hepatitis B in Children. Semin Liver Dis 2003; 23:59-68.

Chan HLY, Hui AY, Wong ML, Tse AML, Hung LCT, Wong VWS, and Sung JJ. Genotype C hepatitis B virus infection is associated with an increased risk of hepatocellular carcinoma. Gut 2004; 53:1494 –1498.

Chang MH, Chen CJ, Lai MS, Hsu HM, Wu TC, Kong MS, Liang DC, Shau WY, and Chen DS. Universal hepatitis B vaccination in Taiwan and the incidence of hepatocellular carcinoma in children. Taiwan Childhood Hepatoma Study Group. N Engl J Med 1997; 336:1855–1859.

Chien RN, Yeh CT, Tsai SL, Chu CM, and Liaw YF. Determinants for sustained HBeAg response to lamivudine therapy. Hepatology 2003;38: 1267–1273.

Chu CJ, Hussain M, and Lok AS. Hepatitis B virus genotype B is associated with earlier HBeAg seroconversion compared with hepatitis B virus genotype C. Gastroenterology 2002; 122:1756 –1762.

Chu CJ, Keeffe EB, Han SH, Perrillo RP, Min AD, Soldevila-Pico C, Carey W, Brown RS Jr, Luketic VA, Terrault N, and Lok AS. Hepatitis B virus genotypes in the United States: results of a nationwide study. Gastroenterology 2003; 125:444–451.

Dane DS, Cameron CH, and Briggs M.Virus-like particles in the serum of patients with Australia-antigen associated hepatitis. Lancet 1970; 1:695–698.

Ding X, Mizokami M, Yao G, Xu B, Orito E, Ueda R, and Nakanishi M. Hepatitis B virus genotype distribution among chronic hepatitis B virus carriers in Shanghai, China. Intervirology 2001; 44:43–47.

Domingo E, Sabo D, Taniguchi T, and Weissmann C. Nucleotide sequence heterogeneity of an RNA phage population. Cell 1978; 13:735–44.

Erhardt A, Reineke U, Blondin D, Gerlich WH, Adams O, Heintges T, Niederau C, and Haussinger D. Mutations of the core promoter and response to interferon treatment in chronic replicative hepatitis B. Hepatology 2000; 31:716–725.

Fattovich G. Natural History and Prognosis of Hepatitis B. Semin Liver Dis 2003; 23:47-58.

Feld J, Lee JY, and Locarnini S. New Targets and Possible New Therapeutic Approaches in the Chemotherapy of Chronic Hepatitis B. Hepatology 2003; 38:545-553.

Fung SK, and Lok AS. Hepatitis B virus genotypes: do they play a role in the outcome of HBV infection? Hepatology 2004; 40:790–792.

Fung SK, Wong F, Hussain M, and Lok ASF. Sustained response after a 2-year course of lamivudine treatment of hepatitis B e antigen-negative chronic hepatitis B. J Viral Hepat 2004; 11:432-438.

Garfein RS, Bower WA, Loney CM, Hutin YJF, Xia GL, Jawanda J, Groom AV, Nainan OV, Murphy JS, and Bell BP. Factors associated with fulminant liver failure during an outbreak among injection drug users with acute hepatitis B. Hepatology 2004; 40:865–873.

Gupta M, Song P, Yates CR, and Meibohm B. Real-time PCR-based genotyping assay for CXCR2 polymorphisms. Clinica Chimica Acta 2004; 341:93–100.

Hadziyannis SJ, Papatheodoridis GV, and Vassilopoulos D. Treatment of HBeAg–Negative Chronic Hepatitis B. Semin Liver Dis 2003; 23:81-88.

Hadziyannis SJ, Vassilopoulos D. Hepatitis B e antigen-negative chronic hepatitis B. Hepatology 2001; 34:617–624.

Hamann L, Hamprecht A, Gomma A, and Schumann RR. Rapid and inexpensive real-time PCR for genotyping functional polymorphisms within the Toll-like receptor -2, -4, and -9 genes. J Immunol Methods 2004; 285:281– 291.

Hannoun C, Horal P, and Lindh M. Long-term mutation rates in the hepatitis B virus genome. J Gen Virol 2000; 81:75–83.

Holland PM, Abramson RD, Watson R, and Gelfand DH. Detection of specific polymerase chain reaction product by utilizing the 5 3 exonuclease activity of Thermus aquaticus. Proc Natl Acad Sci USA 1991; 88:7276-7280.

Janssen HLA, Senturk H, Zeuzem S, Akarca U, Cakaloglu Y, Simon K, et al. Peginterferon alfa-2B and lamivudine combination therapy compared with peginterferon alfa-2B for chronic HBeAg-positive hepatitis B: a randomized controlled trial in 307 patients. Hepatology 2003; 38:1323.

Janssen HLA, van Zonneveld M, Senturk H, Zeuzem S, Akarca US, Cakaloglu Y, Simon C, So TMK, Gerken G, de Man RA, Niesters HGM, Zondervan P, Hansen B, and Schalm SW. Pegylated interferon alfa-2b alone or in combination with lamivudine for HBeAg-positive chronic hepatitis B: a randomised trial. Lancet 2005; 365: 123–29.

Kane M. Global programme for control of hepatitis B infection.Vaccine 1995; 13(Suppl 1):S47–S49.

Kao JH, and Chen DS.（Correspondence）HBV Genotypes and Outcome of HBV Infection. Hepatology 2005; 41:216.

Kao JH, Chen PJ, Lai MY, and Chen DS. Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitis B. Gastroenterology 2000; 118:554 –559.

Kao JH. Hepatitis B viral genotypes: clinical relevance and molecular characteristics. J Gastroenterol Hepatol 2002; 17:643– 650.

Kao JH. Hepatitis B virus genotypes and hepatocellular carcinoma in Taiwan. Intervirology 2003; 46:400–407.

Kao JH, Liu CJ, and Chen DS. Hepatitis B viral genotypes and lamivudine resistance. J Hepatol 2002; 36:303–304.

Kao JH, Wu NH, Chen PJ, Lai MY, and Chen DS. Hepatitis B genotypes and the response to interferon therapy. J Hepatol 2000; 33:998 –1002.

Kirschberg O, Schu¨ ttler C, Repp R, and Schaefer S. A multiplex-PCR to identify hepatitis B virus –genotypes A-F. J Clin Virol 2004; 29:39-43.

Krajden M, Comanor L, Rifkin O, Grigoriew A, Minor JM, and Kapke GF. Assessment of Hepatitis B Virus DNA Stability in Serum by the Chiron Quantiplex Branched-DNA Assay. J Clin Microbiol 1998; 36:382–386.

Kramvis A, and Kew M, and Franc¸ois G. Hepatitis B virus genotypes. Vaccine 2005; 23:2409–2423.

Lee CM, Chen CH, Lu SN, Tung HD, Chou WJ, Wang JH, Chen TM, Hung CH, Huang CC, and Chen WJ. Prevalence and clinical implications of hepatitis B virus genotypes in Southern Taiwan. Scand J Gastroenterol 2003; 38:95–101.

Lee DH, Li L, Andrus L, and Prince AM. Stabilized viral nucleic acids in plasmid as an alternative shipping method for NAT. Transfusion 2002; 42:409-413.

Liaw YF. Results of lamivudine trials in Asia. J Hepatol 2003; 39:S111–S115.

Lindh M, Andersson AS, and Gusdal A. Genotypes, nt 1858 variants, and geographic origin of hepatitis B virus—large-scale analysis using a new genotyping method. J Infect Dis 1997; 175:1285–1293.

Lindh M, Gonzalez JE, Norkrans G, and Horal P. Genotyping of hepatitis B virus by restrixtion pattern analysis of a pre-S amplicon. J Virol Methods 1998; 72:163-174.

Lindh M, Hannoun C, Dhillon AP, Norkrans G, and Horal P. Core promoter mutations and genotypes in relation to viral replication and liver damage in East Asian hepatitis B virus carriers. J Infect Dis 1999; 179:775–782.

Locarnini S, McMillan J, and Bartholomeusz A. The Hepatitis B Virus and Common Mutants. Semin Liver Dis 2003; 23:5-20.

Mackay IM. Real-time PCR in the microbiology laboratory. Clin Microbiol Infect 2004; 10:190–212.

Magnius LO, and Norder H. Subtypes, genotypes and molecular epidemiology of the hepatitis B virus as reflected by sequence variability of the S-gene. Intervirology 1995; 38:24–34.

McCormack GP, and Clewley JP. The application of molecular phylogenetics to the analysis of viral genome diversity and evolution. Rev Med Virol 2002; 12:221–238.

McMahon BJ, Alward WLM, Hall DB, Heyward WL, Bender TR, Francis DP, and 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.

Mizokami M, Orito E, Ohba K, Ikeo K, Lau JY, and Gojobori T. Constrained evolution with respect to gene overlap of hepatitis B virus. J Mol Evol 1997; 44(Suppl 1): S83-S90.

Morrison TB, Weis JJ, and Wittwer CT. Quantification of low copy transcripts by continuous SYBR Green I monitoring during amplification. BioTechniques 1998, 24:954-958, 960, 962.

Naito H, Hayashi S, and Abe K. Rapid and specific genotyping system for hepatitis B virus corresponding to six major genotypes by PCR using typespecific primers. J Clin Microbiol 2001; 39:362–364.

Naumann H, Schaefer S, Yoshida CF, Gaspar AM, Repp R, Gerlich WH. Identification of a new hepatitis B virus (HBV) genotype from Brazil that expresses HBV surface antigen subtype adw4. J Gen Virol 1993; 74: 1627–1632.

Ni YH, Chang MH, Wang KJ, Hsu HY, Chen HL, Kao JH, et al. Clinical relevance of hepatitis B virus genotype in children with chronic infection and hepatocellular carcinoma. Gastroenterology 2004; 127:1733–1738.

Niederau C, Heintges T, Lange S, Goldmann G, Niederau CM, Mohr L, and Haussinger D. Long-term follow-up of HBeAg-positive patients treated with interferon alfa for chronic hepatitis B. N Engl J Med 1996 ;334:1422–7.

Norder H, Courouce AM, and Magnius LO. Complete genomes, phylogenetic relatedness, and structural proteins of six strains of hepatitis B virus, four of which represent two genotypes. Virology 1994; 198:489-503.

Norder H, Hammas B, Lee SD, et al. Genetic relatedness of hepatitis B viral strains of diverse geographical origin and natural variations in the primary structure of the surface antigen. J Gen Virol 1993 ;74:1341–8.

Okamoto H, Tsuda F, Sakugawa H, et al. Typing hepatitis B virus by homology in nucleotide sequence: comparison of surface antigen subtypes. J Gen Virol 1988; 69:2575–83.

Orito E, Mizokami M, Sakugawa H, Michitaka K, Ishikawa K, Ichida T, et al. A case-control study for clinical and molecular biological differences between hepatitis B viruses of genotypes B and C. Japan HBV Genotype Research Group. Hepatology 2001; 33:218 –223.

Osiowy C, and Giles E. Evaluation of the INNO-LiPA HBV Genotyping Assay for Determination of Hepatitis B Virus Genotype J Clin Microbiol 2003; 41:5473–5477.

Owiredu WK, Kramvis A, and Kew MC. Hepatitis B virus DNA in serum of healthy black African adults positive for hepatitis B surface antibody alone: possible association with recombination between genotypes A and D. J Med Virol 2001; 64:441–454.

Papatheodoridis GV, Dimou E, and Papadimitropoulos V. Nucleoside Analogues for Chronic Hepatitis B: Antiviral Efficacy and Viral Resistance. Am J Gastroenterol 2002; 97:1618–1628.

Park SG, Kim Y, Park E, Ryu1 HM, and Jung G. Fidelity of hepatitis B virus polymerase. Eur J Biochem 2003; 270:2929–2936.

Payungporn S, Tangkijvanich P, Jantaradsamee P, Theamboonlers A, and Poovorawan Y. Simultaneous quantitation and genotyping of hepatitis B virus by real-time PCR and melting curve analysisJournal of Virological Methods 2004; 120:131–140.

Pocsai Z, To´th Z, Paragh G, Sze´les G, and A´da´ny R. Rapid genotyping of paraoxonase 55 and 192 mutations by melting point analysis using real time PCR technology. Clinica Chimica Acta 2003; 332:31–36.

Rehermann B. Immune Responses in Hepatitis B Virus Infection. Semin Liver Dis 2003; 23:21-37.

Sanchez-Tapias JM, Costa J, Mas A, Bruguera M, and Rodes J. Influence of hepatitis B virus genotype on the long-term outcome of chronic hepatitis B in western patients. Gastroenterology 2002; 123:1848 –1856.

Seeger C, and Mason WS. Hepatitis B Virus Biology. Microbiol Mol Biol Rev 2000; 64:51–68.

Shikata T, Karasawa T, Abe K, Uzawa T, Suzuki H, Oda T, Imai M, Mayumi M, and Moritsugu Y. Hepatitis B e antigen and infectivity of hepatitis B virus. J Infect Dis 1977 ;136:571–576.

Singh M, Dicaire A, Wakil AE, Luscombe C, and Sacks SL. Quantitation of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in the liver of HBV-infected patients by LightCyclerTM real-time PCR. J Virol Methods 2004; 118:159–167.

Steinhauer DA, and Holland JJ. Direct method for quantitation of extreme polymerase error frequencies at selected single base sites in viral RNA. J Virol 1986; 57:219–28.

Stevens CE, Beasley RP, Tsui J, and Lee WC. Vertical transmission of hepatitis B antigen in Taiwan. N Engl J Med 1975; 292:771–774.

Sugauchi F, Orito E, Ichida T, et al. Epidemiologic and Virologic Characteristics of Hepatitis B Virus Genotype B Having the Recombination With Genotype C. Gastroenterology 2003; 124:925–932.

Sugauchi F, Orito E, Ichida T, et al. Hepatitis B virus of genotype B with or without recombination with genotype C over the precore region plus the core gene. J Virol 2002; 76:5985–5992.

Sumi H, Yokosuka O, Seki N, Arai M, Imazeki F, Kurihara T, et al. Influence of hepatitis B virus genotypes on the progression of chronic type B liver disease. Hepatology 2003; 37:19 –26.

Summers J, and Mason WS. Replication of the genome of a hepatitis B–like virus by reverse transcription of an RNA intermediate. Cell 1982; 29:403–415.

Suzuki S, Sugauchi F, Orito E, Kato H, Usuda S, Siransy L, Arita I, Sakamoto Y, Yoshihara N, El-Gohary A, Ueda R, and Mizokami M. Distribution of hepatitis B virus (HBV) genotypes among HBV carriers in the cote d’Ivoire: complete genome sequence and phylogenetic relatedness of HBV genotype E. J Med Virol 2003; 69:459–465.

Szmuness W, Stevens CE, Harley EJ, Zang EA, Oleszko WR, William DC, Sadovsky R, Morrison JM, and Kellner A. Hepatitis B vaccine: demonstration of efficacy in a controlled clinical trial in a high-risk population in the United States. N Engl J Med 1980; 303:833–841.

Tassopoulos NC, Papaevangelou GJ, Sjogren MH, et al. Natural history of acute hepatitis B surface antigen–positive hepatitis in Greek adults. Gastroenterology 1987; 92:1844–1850.

Teles SA, Martins RM, Vanderborght B, Stuyver L, Gaspar AM, Treichel U, Meyer zum Bu schenfelde KH, Dienes HP, and Gerken G. Receptor-mediated entry of hepatitis B virus particles into liver cells. Arch Virol 1997; 142:493-498.

Usuda S, Okamoto H, Iwanari H, et al. Serological detection of hepatitis B virus genotypes by ELISA with monoclonal antibodies to type-specific epitopes in the preS2-region product. J Virol Methods 1999; 80:97–112.

Usuda S, Okamoto H, Tanaka T, et al. Differentiation of hepatitis B virus genotypes D and E by ELISA using monoclonal antibodies to epitopes on the preS2-region product. J Virol Methods 2000; 87:81–89.

Wai CT, Chu CJ, Hussain M, and Lok AS. HBV genotype B is associated with better response to interferon therapy in HBeAg(+) chronic hepatitis than genotype C. Hepatology 2002; 36:1425–1430.

Wai CT, Fontana RJ, Polson J, Hussain M, Shakil AO, Han SHB, et al. Clinical outcome and virological characteristics of hepatitis B related acute liver failure in the United States. J Viral Hepat 2005; 12:192-198.

Westland CE, Delaney WT, Yang H, Chen SS, Marcellin P, Hadziyannis S, et al. Hepatitis B virus genotypes and virologic response in 694 patients in phase III studies of adefovir dipivoxil. Gastroenterology 2003; 125:107–116.

Westland CE, Yang H, Delaney IV WE, Gibbs CS, Miller MD, Wulfsohn M, Fry J, Brosgart CL, and Xiong S. Week 48 Resistance Surveillance in Two Phase 3 Clinical Studies of Adefovir Dipivoxil for Chronic Hepatitis B. Hepatology 2003; 38:96-103.

Wilhelm J, and Pingoud A. Real-Time Polymerase Chain Reaction. ChemBioChem 2003; 4:1120-1128.

Wittwer CT, Ririe KM, Andrew RV, David DA, Gundry RA, and Balis UJ. The LightCycler™: A Microvolume Multisample Fluorimeter with Rapid Temperature Control BioTechniques 1997; 22:176-181.

Yeh SH, Tsai1 CY, Kao JH, Liu CJ, Kuo TJ, Lin MW, Huang WL, Lu SF, Jih1 J, Chen DS, and Chen PJ. Quantification and genotyping of hepatitis B virus in a single reaction by real-time PCR and melting curve analysis. J Hepatol 2004; 41:659–666.

Yoshida CF. Hepatitis B virus: genotypes and subtypes in Brazilian hemodialysis patients. Artif Organs 1999; 23:1074–1078.

Yuen MF, and Lai CL. Treatment of chronic hepatitis B. Lancet Infect Dis 2001; 1: 232–41.

Yuen MF, Wong DK, Sablon E, Yuan HJ, Sum SM, Hui CK, et al. Hepatitis B virus genotypes B and C do not affect the antiviral response to lamivudine. Antivir Ther 2003; 8:531–534.

Zeng GB, Wen S-J, Wang Z-H, Yan L, Sun J, and Hou J-L. A novel hepatitis B virus genotyping system by using restriction fragment length polymorphism patterns of S gene amplicons. World J Gastroenterol 2004; 10:3132-3136.

Zhou L, Myers AN, Vandersteen JG, Wang L, and Wittwer CT. Closed-Tube Genotyping with Unlabeled Oligonucleotide Probes and a Saturating DNA Dye.Clin Chem 2004; 50:1328-1335.