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研究生：	劉紋君 Liu, Wen-Chun
論文名稱：	B 型肝炎病毒變異株與其臨床意義之相關研究─針對病毒基因型、病毒演化及病毒基因突變率與病程之相關性進行探討 The study of hepatitis B virus variants and their clinical significance─ focusing on viral genotypes, viral evolution, and the relationship between mutation rate of viral genome and disease progression
指導教授：	張定宗 Chang, Ting-Tsung
學位類別：	博士 Doctor
系所名稱：	醫學院 - 基礎醫學研究所 Institute of Basic Medical Sciences
論文出版年：	2008
畢業學年度：	96
語文別：	英文
論文頁數：	126
中文關鍵詞：	即時PCR 、熔解曲線分析、基因亞型、血清轉換、突變率、基因型、B型肝炎病毒
外文關鍵詞：	subgenotype, melting curve analysis, real-time PCR, genotype, Hepatitis B virus, seroconversion, mutation rate
相關次數：	點閱：263 下載：4
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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。大部分的B型肝炎病毒基因型具有地理上分布的獨特性，並且病毒基因型會影響慢性B型肝炎病臨床的表現。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 與 BEF兩群組）。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429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。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反應能夠同時定性與定量，定量敏感度為103~1013 copies/mL，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。 (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。 (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92.5%。 (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90.6%及47.8%。簡而言之，我們成功地設立了有效的HBV基因型分型方法，精準地鑑定出A~G七型的HBV基因型，具有高偵測敏感度、同時能定量病毒及高準確率的特性。此外，B型肝炎病毒基因型B是分布於亞洲最主要的基因型之ㄧ，根據演化樹譜分析，我們發現基因型B可以在被細分為基因亞型B1到B5，其中基因亞型B1、B3、B4與B5具有地理上的獨特性，分別只分布在日本、印尼、越南以及菲律賓，此五個基因亞型的病毒序列也具有相當特異性的部份，並且B2、B3、B4以及B5的Core基因與基因型C的Core基因發生序列重組。我們利用基因亞型的地理位置分佈、基因亞型間的序列差異度勾畫出B基因亞型的演化地理分布關係網絡，提供B基因亞型在亞洲地理上的演化關係。病毒的突變以及演化的速率與臨床病程有關，根據病人的臨床的表現，本研究納入53位臨床未用藥的病人，將病人依長達一年以上追蹤期間病毒量的起伏，分為二種型態，分別是穩定型 (stationary pattern)以及起伏型(fluctuated pattern)。其中起伏型的類別依照追蹤期間是否達到血清轉換，再被細分為血清轉換組與無血清轉換二組。我們依此分類，比較各型病毒序列變異以及突變速率的差異。根據結果顯示，起伏型的病毒突變速率比穩定型快，且在病毒核心區域以及核心調控區域達到統計上的意義(P<0.05)。與血清轉換組比較，對於未達到血清轉換的病人而言，在病毒量急劇上升時期病毒核心蛋白的突變率，有意義地高於隨後病毒量下降時期的突變率。在絕大多數核心蛋白的突變，起伏型與穩定型差異是有意義的。同時這些突變包含許多hot-spots且已被證實與逃脫免疫的機制有關。這些發現將可能有助於未來進一步闡釋宿主與病毒間的相互作用機制。

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. 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, 429 clinical samples which showed positive HBsAg and HBV DNA were examined. 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. (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. (3) The genotype-specific primers (N1/ABFG & N2/CDE) used in multiplex PCR succeeded in differentiating most samples with 92.5% accuracy. (4) In the previous research, RFLP (1998) and genotype-specific primers (2001) assays could reach 90.6% and 47.8% of accuracy, respectively. 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. 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. In addition, the genotype B of HBV is the major genotype in Asia. We found the genotype B could be classified into five subgenotypes, and named in HBV/B1, B2, B3, B4 and B5 according to phylogenetic tree and sequence diversity. The subgenotypes B1, B3, B4 and B5 had geographic uniqueness and were distinctively distributed in Japan, Indonesia, Vietnam and Philippine. Some nucleotide and amino acid sequences of the five subgenotypes were also distinctive. Furthermore, the core gene of subgenotypes B2, B3, B4 and B5 recombined with that of genotype C. We clearly drew a phylogeography network to point out the evolution history of HBV genotype B in Asia by phylogenetic tree, geography and diversity. The evolution rate was correlated with mutation rate of virus. On the other hand, the mutation and evolution rates of the virus were correlated with clinical process. In this study, we also enrolled 53 patients and classified them into two patterns according to their viral kinetics during more than one year follow-up without antiviral treatment, i.e. stationary and fluctuated viral kinetic patterns. The patients with fluctuated pattern were further categorized into seroconversion and non-seroconversion groups depending on whether achieving seroconversion or not during the follow-up period. According to the classification, we compared viral nucleotide and amino acid sequence substitutions and their mutation rates among different viral kinetic patterns. In the results, the mutation rate of fluctuated pattern was higher than that of stationary pattern, especially in the core gene and regulatory element of core (P<0.05). As comparing with seroconversion group, non-seroconversion group had significantly higher mutation rate of viral core protein during viral DNA breakthrough than that during viral decline later. The majority of core protein mutations, which contained many hot-spots correlated with escaping immune response, were significantly different between fluctuated pattern and stationary pattern. These findings will be probably helpful to further illustrate the mechanism of host-virus interaction in the future.

Contents
Abstract (in Chinese)------------------------------------------------------------I
Abstract (in English)----------------------------------------------------------III
Index----------------------------------------------------------------------------VI
Figure / Table index------------------------------------------------------------X
Introduction----------------------------------------------------------------------1
Aim and study strategy--------------------------------------------------------12
Materials and methods--------------------------------------------------------13
1. To develop an efficient PCR-based method with quantification and genotyping of HBV genotypes A to G in one-tube reaction.                         13
1.1	Study subjects and samples                                       13
1.2 Principle of real-time PCR genotyping by melting curve analysis 13
1.3 Real-time PCR amplification of HBV using Lightcycler               14
1.4 Ouantification of HBV DNA by real-time PCR                      15
1.5 Alternative genotyping of HBV genotypes A, C, D, and G by type-specific multiplex PCR                                                 15
1.6 Direct sequencing analyses                                       16
1.7 Statistical methods                                              16
2. To develop an efficient multiplex PCR method to differentiate HBV genotypes A to G in one step.                                                 18
2.1	Patients                                                       18
2.2	Principles of multiplex PCR genotyping                            18
2.3	HBV genotyping by multiplex PCR                                19
2.4	Direct sequencing analyses                                       19
3. To analyze the phylogeography of the subgenotypes of HBV by the phylogenetic analysis and HBV distribution                            21
3.1 HBV Isolates From Patients and GenBank                          21
3.2 DNA extraction, amplification, and sequencing                      21
3.3 Phylogenetic analysis.                                           22
4. To compare mutation rate, nucleotide and amino acid sequence changes and protein structure of HBV in natural course between patients with stationary and fluctuated viral kinetic patterns                                  24
4.1 Patients                                                       24
4.2 Viral kinetic patterns of HBV                                     24
4.3 DNA extraction, amplification, and sequencing                      25
4.4  HBV genotyping and phylogenic analysis                          25
4.5. Protein analysis                                                26
4.6 Statistical analysis                                              26
Results----------------------------------------------------------------------------28
1.	 To develop an efficient PCR-based method with quantification and genotyping of HBV genotypes A to G in one-tube reaction.             28
1.1 Quantification of HBV by set 1 amplicon (ACDG/BEF set)            28
1.2 Genotyping by melting curve analysis (MCA)                       28
1.3 Typing of genotypes A, C, D, and G by multiplex PCR                29
1.4 Detection of HBV mixed infections with melting curve analysis         29
2. To develop an efficient multiplex PCR method to differentiate HBV genotypes A to G in one step.                                                 31
2.1 Differentiation of 7 genotypes by one step multiplex PCR with type specific primers                                                       31
2.2 Detection of HBV mixed infections with multiplex PCR         31
2.3 Accuracy of the genotyping methods                               31
3. To analyze the phylogeography of the subgenotypes of HBV by the phylogenetic analysis and HBV distribution                            32
3.1 Phylogenetic analysis and geographical distribution with five Subgenotypes of Genotype B.                                                 32
3.2	Nucleotide divergence among HBV/B1-B5 and other genotypes         33
3.3 Comparison of the nucleotide sequences of cis-acting elements of HBV/B1-B5 and other genotypes                                   34
3.4 Comparison of amino acid sequences of HBV/B1-B5 and other genotypes 34
4.	 To compare mutation rate, nucleotide and amino acid sequence changes and protein structure of HBV in natural course between patients with stationary and fluctuated viral kinetic patterns                                  36
4.1 Comparison of characteristics of patients with stationary and fluctuated viral kinetic patterns                                            36
4.2 Comparison of HBV mutation rate of three phases between stationary and fluctuated viral kinetic patterns                                   36
4.3 Comparing mutation rates of nucleotides and amino acids in different phases between stationary and fluctuated viral kinetic patterns         38
4.4 Comparison of HBV mutation rate of three phases between seroconversion and non-seroconversion groups in fluctuated viral kinetic pattern.      38
4.5 Hot spot expression of HBV core protein among patients with different viral kinetic pattern                                             39
4.6 Structure characteristics of HBV core protein in patients with stationary and fluctuated viral kinetic patterns                               40
4.7 Construction changes at the viral peak to benefit the viral exist.        40
Discussion------------------------------------------------------------------------42
Reference-------------------------------------------------------------------------55
Figures--------------------------------------------------------------------------67
Tables-----------------------------------------------------------------------------93
Author-----------------------------------------------------------------------------------------125

Figure / Table contents
Figure 1. The primers and hybridization probes on melting curve analysis------67
Figure 2. Quantification of the virus by real-time PCR with Set 1-----------------69
Figure 3. The representative results for melting curve analysis of clinical samples with genotypes A to G ----------------------------------------------------------71
Figure 4. The determination of HBV mixed infection by the melting curve analysis ----------------------------------------------------------------------------73
Figure 5. The electrophoresis results of multiplex PCR for identifying genotype A, C, D and G --------------------------------------------------------------------74
Figure 6. Strategy for genotyping of genotype A to G by multiplex PCR using type-specific primers -----------------------------------------------------------75
Figure 7. The electrophoresis results of multiplex PCR for identifying genotypes A to G.-----------------------------------------------------------------------------76
Figure 8. Complete genome and preC plus core gene of neighbor-joining phylogenetic HBV tree ---------------------------------------------------------78
Figure 9. Minimum spanning network of complete genome sequences of HBV subgenotypes B1-B5--------------------------------------------------------------82
Figure 10. Characteristic presentation of stationary and fluctuated viral kinetic patterns of HBV -----------------------------------------------------------------83
Figure 11. Comparing mutation rates of nucleotide and amino acids in different phases between stationary and fluctuated viral kinetic patterns------85
Figure 12. Comparing of mutation rate from baseline to final point between seroconversion group and non-seroconversion group in fluctuated viral kinetic pattern ------------------------------------------------------------87
Figure 13. Comparing of nucleotide mutation sites of 24 complete HBV genomes from basline to final point ----------------------------------------------------89
Figure 14. Comparing mutation rates of core gene in different phases between stationary and fluctuated viral kinetic patterns including seroconversion and non-seroconversion groups --------------------------90
Figure 15. Mutated hot spots of core protein from baseline to final point among different viral kinetic patterns -----------------------------------------------91
Figure 16. Comparing consistence of mutated amino acids of core protein during different phases to those at final points between seroconversion and non-seroconversion groups ---------------------------------------------------92

Table 1. Sequence variations of A–G genotypes at signature genetic polymorphisms of three sets of amplicons in 367 full-length viral isolates-------------------------------------------------------------------------------93
Table 2. PCR primers and probes in the amplicons used for HBV quantification and genotyping of genotype A to G with Real-time PCR and Two-step Melting Curve Analysis---------------------------------------------------------94
Table 3. Type specific PCR primers in the amplicon used for typing of HBV genotype A, C, D and G by multiplex PCR -------------------------------96
Table 4. Sequence variations of A–G genotypes at signature genetic polymorphisms of amplicons designed for two-step meltimg curve analysis in 367 full-length viral isolates -----------------------------------97
Table 5. Comparing the accuracy of HBV genotyping method -----------------99
Table 6. The type specific PCR primers in the amplicon used for HBV genotyping of genotypes A to G by multiplex PCR ---------------------100
Table 7. Sequence variations of A–G genotypes at signature genetic polymorphisms of amplicons designed for multiplex PCR pcr in 367 full-length viral isolates -------------------------------------------------------103
Table  8. Geographic distribution of 479 HBV isolates according to genotypes A to G -------------------------------------------------------------------------------106
Table  9. Mean percentage of nucleotide divergence over the complete genome and core gene (containing precore region) among 479 human HBV isolates with a woolly monkey hepatitis virus isolate as the out group------------------------------------------------------------------------------107
Table 10. Comparison of the nucleic acid sequences of the cis-acting elements of HBV/B1-B5 isolates with other HBV genotypes -------------------------108
Table 11. Characteristic mutations within the cis-acting regulatory elements found in the five subgenotype of HBV/B ---------------------------------111
Table 12. Comparison of amino acid residues of four ORFs of HBV/B1–B5 isolates with other HBV genotypes ----------------------------------------113
Table 13. Characteristic mutations within amino acids of each HBV/B subgenotype---------------------------------------------------------------------117
Table 14. Genograpgic distribution of 168 HBV/B isolates according to their subgenotypes --------------------------------------------------------------------119
Table 15. The primers used for full-length and second-round PCR and for sequencing -----------------------------------------------------------------------120
Table 16. Comparison of characteristics of patients with stationary and fluctuated viral kinetic patterns --------------------------------------------121
Table 17. The list of core protein hotspots among patients with different viral kinetic pattern ------------------------------------------------------------------122
                                    

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