核苷类似物干预下慢性乙型肝炎患者临床疗效及HBV变异与进化规律的研究
|
摘要
慢性乙型肝炎(CHB)威胁全球人类健康,其中1/3以上的慢性感染者在中国。抗病毒治疗是慢性乙型肝炎的核心治疗措施。核苷类似物(NUC)是目前抗病毒治疗的主流药物。同时,核苷类似物在使用过程中遇到一系列难以回答的问题,例如:核苷类似物耐药的预防和处理;耐药出现后挽救治疗方案如何选择(联合或是序贯);停药时机的把握等。我们注意到,核苷类似物的耐药问题是上述问题的核心。目前核苷类似物的使用也主要把握两点:一是充分发挥药物的抗病毒功效,另一方面则是尽量避免耐药的发生。然而目前临床使用核苷类抗病毒药物缺乏必要的实验室依据,同时对治疗过程中病毒变异及进化模式和耐药发生机制尚未阐明,这是制约核苷类似物科学合理应用的关键问题。
对已经处于慢性HBV感染的大量人群,尤其是已逐渐进入免疫清除期即发病阶段的患者,以持久的抑制HBV复制为首要目标的核苷类似物(NUCs)治疗成为临床治疗的主要手段。通过大量循证医学证据证实,NUCs可以显著减缓和防止乙型肝炎进展为肝硬化、肝衰竭和肝细胞癌,从而成为目前的主流疗法并广泛使用。大范围NUCs的干预改变了患者体内HBV的多样性、进化速率及演变方向。病毒在药物压力下形成耐药株,耐药株的流行及传播将会对乙型肝炎的流行和防治产生深远影响。核苷类似物对于病毒而言是一种新的环境,在药物作用压力下HBV逆转录酶区(RT区)整体上进化规律如何?RT区是否受到药物正选择压力的作用?我国CHB人群主要为基因B型及C型,核苷类似物疗法对于这两种不同基因型的选择压力是否有所不同?这些问题目前尚缺乏大样本的研究。
已有的关于NUCs耐药的研究大多数集中在RT区A至D结构域内,目前常规使用的几种NUCs的耐药位点都已比较明确。HBV基因组50%的基因组为重叠编码区,RT区核苷酸位点的突变同样可以导致表面抗原S区编码氨基酸的改变。由于HBV是以全长序列为遗传单位进行复制和传播,同时也受到宿主免疫及药物压力的选择。宿主免疫压力主要选择位点为S区和C区,而NUCs则主要为RT区。因此,NUCs选择株的变异必然是全基因组范围并且相互关联的。基于此,对NUCs治疗过程中HBV其他编码区(尤其是免疫原性较强的C区和S区)的动态变化的监测对于认识NUCs耐药相关的肝炎突发的机制具有重要意义。同时,由于HBV具有准种特性,宿主免疫和抗病毒药物的作用可能使得该群体中出现对选择压力具有生存优势的变异株,因此阐明HBV准种的变异和进化模式对于CHB的治疗和管理具有指导价值。以往有许多的针对NUCs治疗患者HBV RT区进行的准种方面的研究。然而这些研究均只关注了NUCs作用的靶区域即RT区,而没有对HBV全长进行研究。而新近的对HBV全基因组序列的研究均采用的是PCR直接测序法扩增HBV全长序列,或通过几个扩增片段拼接得到全长序列,而没有采用克隆的方法。因此,NUCs干预下HBV准种全长序列变异及进化模式目前仍未明确。
为探讨上述问题,本研究收集了212例LAM耐药而后加用ADV治疗的患者,对不同基因型患者LAM耐药变异模式的差异以及ADV+LAM联合疗法对于不同类型LAM耐药人群的疗效进行了分析。在大样本(1 033例)的接受核苷类似物治疗的患者,采用PCR-直接测序法得到HBV RT区基因序列,进行RT区基因系统进化树构建,利用基于最大似然法的密码子替换模型分别在基因B型及C型序列中检测其可能的正选择作用位点以及差异性位点,以期从整体进化的角度分析RT区基因的进化特征。同时为探索NUCs干预下HBV准种全长序列变异及进化模式,我们纳入了5例核苷类似物序贯/联合治疗的患者以及2例未经治疗但有肝炎发作的患者,采用PCR-克隆测序-种系进化分析方法,对随访过程中的系列血清进行了HBV准种全长序列变异及进化规律的分析。
主要结果:
一、基因C型患者发生LAM突破的时限长于B型患者(P=0.036)。LAM+ADV联合治疗3个月后,所有患者平均HBV DNA水平较LAM突破时显著下降(P<0.001)。180例(84.9%)患者ALT复常,15例(11.6%)患者出现HBeAg阴转。基因B、C型患者联合治疗后HBV DNA水平、ALT复常率、HBeAg阴转率无明显差异。
二、212例患者中共计193例检测出rt204位点变异。L180M与M204V联合变异的频率显著高于M204I(76/80 vs. 28/113,P<0.001)。C型患者检出M204I+L180M变异的频率高于B型患者(P=0.002),而M204V/I+V207L变异则在B型患者中比例更高(P=0.005)。M204V+L180M、M204V/I+229及M204V/I+S213T变异在基因B、C型患者中的分布无明显差异。42例(19.8%)患者出现多位点(3个及以上位点)耐药变异。
三、M204V/I变异联合及未联合rt180、rt229、rt213位点变异组在LAM治疗基线、LAM突破及联合治疗后的HBV DNA水平、ALT水平及复常率上均无明显差异。M204V/I未合并V207L变异组联合治疗后ALT复常率高于M204V/I合并V207L变异组(P=0.001),而HBV DNA水平无明显差异。
四、基因B型RT区序列平均非同义(dN)/同义突变率(dS)比值ω=0.26,正选择位点为rt204位及rt222位氨基酸。基因C型RT区序列平均ω=0.19,正选择位点为rt180位及rt204位氨基酸。
五、基因B型及C型RT区序列有六个位点存在选择压力的差异,分别为:rt182 (P=0.08)、rt209(P=0.09)、rt211(P<0.001)、rt226(P=0.04)、rt235(P=0.08)以及rt238(P=0.02)。
六、3例LAM初治耐药而后换用其他NUCs治疗的患者HBV全基因组核苷酸及各编码区氨基酸异质性在出现LAM病毒学突破时均降至最低点,而在拯救治疗后又重新增高。接受ADV-替比夫定(LDT)序贯治疗的两例患者基因组异质性的变化则各有其特点。
七、2例未治疗组患者在出现肝炎突发时核苷酸及氨基酸异质性均较无症状期升高。
八、HBV基因组各编码区在慢性化感染过程中的遗传异质性的变化趋势各不相同,不同的患者也各有其特征。
九、5例经NUCs治疗的患者均检测出NUC相关耐药位点变异,3例患者出现多药(重)耐药。所有患者治疗前“a”决定簇均未检测出氨基酸改变,而在治疗后4例患者在该区出现了编码氨基酸的改变。未治疗的两例患者大多数的变异均集中位于前C/C区。
十、5例治疗组患者在前S/S区或C区的T细胞或B细胞表位中均出现了伴随RT区变异的编码氨基酸的改变。部分患者在核心启动子区及前C区等也检测到伴随RT区变异的核苷酸突变。
通过对这些结果进行分析并和既往研究结果进行比较,结论如下:
一、LAM+ADV联合疗法对于不同基因型(B型及C型)及不同LAM变异类型的耐药患者均能够取得较好的疗效。
二、基因B型及C型患者LAM补偿耐药位点(L180M及V207L)的分布有一定的差异。LAM初始单药治疗的患者中有一部分可能出现多位点耐药变异,提示对这部分患者初始抗病毒治疗应选择高基因屏障的药物。
三、通过大样本HBV RT区基因序列的进化分析,证实在NUCs干预下RT区基因整体上以净化选择的模式为主,少数位点则呈现为正选择的进化模式。基因B型及C型RT区基因在部分非NUC耐药变异位点上所受到的选择压力有所不同。
四、不同NUC药物治疗方式对HBV全基因组准种的进化方式有影响:采用LAM初治患者在LAM耐药时病毒异质性降低,而在挽救治疗之后异质性又逐渐升高;而ADV突破后换用其他NUC治疗的患者HBV准种则具有不同的进化规律。
五、经NUCs治疗的患者与未治疗但出现肝炎突发的患者HBV准种具有不同的进化及变异模式:未治疗患者在肝炎突发时HBV基因组变化主要集中在前C/C区,而在治疗组患者则未观察到此规律。提示引起NUCs治疗患者与未治疗患者肝炎突发的病毒学机制可能有所不同。
六、经NUCs治疗的患者在出现NUCs耐药时均检测出前S/S区和/或C区表位编码氨基酸的改变,提示HBV RT区变异可伴随有其他编码区表位的变化,使得HBV免疫原性发生改变。
Chronic hepatitis B virus (HBV) infection is a major public health problem worldwide. In recent years, treatment of chronic HBV has been improved, with the availability of nucleoside/nucleotide analogues (NUCs) such as lamivudine (LAM). Unfortunately, the clinical benefit is rarely sustained under long-term treatment due to the selection of lamivudine resistant mutants, which occur at an annual rate of 14-32%. Adefovir (ADV) has an antiviral profile with potent activity against lamivudine-resistant strains, making it useful as a rescue treatment for lamivudine resistant patients. However, suboptimal responses have also been reported after adefovir rescue therapy, and its efficacy for patients with different genotypes and different LAM-resistant mutations remains unclear.
Most studies of HBV drug resistance have focused on analysis of the A to D domain of the HBV DNA polymerase gene after detection of antiviral-resistant HBV mutants, and the drug-resistant mutational pattern that occurs in this region of the gene has been well characterized. The full HBV genome contains four partially overlapping open reading frames (ORFs) and mutations in the reverse transcriptase (RT) domain can affect the amino acid sequence of the surface protein. Previous studies have found that the immune-escape variants probably appear after antiviral-resistant variants emerge; this might be responsible for the exacerbation of chronic hepatitis B in some patients. Based on these findings, dynamic changes within other regions (especially in the immune-targeted surface and core antigen) might also be informative, and comparison of the full genome is necessary.
Like other RNA viruses, HBV shows quasispecies distribution in infected patients. Thus, to permit chronic infection management, the evolution and distribution of HBV should be monitored. These studies about the dynamic evolution of HBV quasispecies during treatment of patients with commonly used NUCs focused on the HBV DNA polymerase gene and did not investigate the mutational pattern of the full HBV genome. Several more recent studies have reported the mutational pattern of the full HBV genome. However, the HBV full genome sequence was examined by direct sequencing, not by cloning. Thus, the evolutionary pattern of complete HBV quasispecies remains unclear.
The present study investigated patterns of lamivudine-resistant mutations in 212 patients with lamivudine-resistance and evaluated the effect of these mutations on the antiviral response to LAM+ADV combination therapy. 1 033 sequences of HBV RT region derived from the patients with NUCs therapy were used to elucidate the evolution pattern and positive selection in HBV population. Meanwhile, we used full genome sequences generated from patient sera to assess the quasispecies evolutionary dynamics of five chronic hepatitis B patients who underwent sequential NUC therapies, as well as of two untreated patients with acute flares.
Main Results
1. The duration for LAM resistance in patients with genotype C was significantly longer than those with genotype B (P=0.036). Mean HBV DNA level after 3 months of LAM+ADV combination therapy was lower than that in LAM resistance (P<0.001). 180 patients (84.9%) achieved ALT normalization and 15 patients (11.6%) achieved HBeAg seroconversion. There was no difference in HBV DNA level, ALT normalization and HBeAg seroconversion rate between patients with genotype B and C.
2. rt204 mutation was detected in 193 patients. L180M was more often found in patients with M204V than those with M204I (76/80 vs. 28/113,P<0.001). M204I+L180M combined mutation presented more in patients with genotype C (P=0.002), and M204V/I+V207L was detected more in genotype B (P=0.005). There was no difference in the incidence of M204V+L180M, M204V/I+229 and M204V/I+S213T mutation between patients with genotype B and C. Multi-mutations were detected in 42 patients (19.8%).
3. The presence of the rt180、rt229、rt213 did not significantly affect viral load reduction and ALT normalization during combination therapy. The proportion of ALT normalization for patients carrying M204V/I combined with V207L was lower than those without V207L (P=0.001).
4. Mean nonsynonymous (dN)/synonymous (dS) ratioωfor genotype B sequences was 0.26; rt204 and rt222 were identified as positively selected sites. Meanωfor genotype C sequences was 0.19; rt204 and rt180 were identified as positively selected sites.
5. Significantly different selection was identified in six codons between genotype B and C sequences, including rt182 (P=0.08), rt209 (P=0.09), rt211 (P<0.001), rt226 (P=0.04), rt235 (P=0.08) and rt238 (P=0.02).
6. In the three treated patients who received lamivudine as initial antiviral therapy, nucleotide polymorphism and nonsynonymous divergence decreased at lamivudine breakthrough but increased after rescue therapies. Conversely, two other treated patients showed distinct change in divergence during adefovir-telbivudine sequential therapies.
7. Untreated subjects exhibited increased polymorphism and divergence in the preC/C region at hepatitis flare.
8. Divergence of different HBV coding regions showed different changing trend in a single host and within different patients.
9. All treated patients showed NUCs resistant mutations, three of which showed multi-drug mutations. Four of the treated patients presented amino acid changes in the“a”determinant during NUC therapy. In untreated patients, most of the variations were located in the preC/C gene.
10. All of the treated subjects showed amino acid changes within the known T-cell or B-cell epitopes in the surface or core antigen, most of which were accompanied by mutations in the RT region. Co-variations in the core promoter, the preC region and the known epitopes of the preS gene, accompanied by RT mutations, were not rare.
From these results, we draw conclusions as follows:
1. LAM and ADV combination therapy is effective for patients with genotype B and C and for patients with different LAM-resistant patterns.
2. There are differences for the LAM-resistant patterns between patients with different genotypes. Patients who receive LAM as initial therapy have risk to develop multi-mutations.
3. Purified evolution prevail HBV RT region for patients who receive NUCs therapy, with a small portion of codons under positive selection. There is difference in selection pressure between patients with genotype B and C.
4. The distribution of genetic variability of HBV shows remarkably different patterns between NUC-treated and untreated subjects and within patients receiving different NUCs therapies.
5. The underlying mechanisms of hepatitis flare differ between NUC-treated and untreated patients.
6. NUC-resistant mutations can cause amino acid changes within epitopes of other HBV regions and that the characteristics of HBV immunogenicity may change when NUC resistance emerges.
对已经处于慢性HBV感染的大量人群,尤其是已逐渐进入免疫清除期即发病阶段的患者,以持久的抑制HBV复制为首要目标的核苷类似物(NUCs)治疗成为临床治疗的主要手段。通过大量循证医学证据证实,NUCs可以显著减缓和防止乙型肝炎进展为肝硬化、肝衰竭和肝细胞癌,从而成为目前的主流疗法并广泛使用。大范围NUCs的干预改变了患者体内HBV的多样性、进化速率及演变方向。病毒在药物压力下形成耐药株,耐药株的流行及传播将会对乙型肝炎的流行和防治产生深远影响。核苷类似物对于病毒而言是一种新的环境,在药物作用压力下HBV逆转录酶区(RT区)整体上进化规律如何?RT区是否受到药物正选择压力的作用?我国CHB人群主要为基因B型及C型,核苷类似物疗法对于这两种不同基因型的选择压力是否有所不同?这些问题目前尚缺乏大样本的研究。
已有的关于NUCs耐药的研究大多数集中在RT区A至D结构域内,目前常规使用的几种NUCs的耐药位点都已比较明确。HBV基因组50%的基因组为重叠编码区,RT区核苷酸位点的突变同样可以导致表面抗原S区编码氨基酸的改变。由于HBV是以全长序列为遗传单位进行复制和传播,同时也受到宿主免疫及药物压力的选择。宿主免疫压力主要选择位点为S区和C区,而NUCs则主要为RT区。因此,NUCs选择株的变异必然是全基因组范围并且相互关联的。基于此,对NUCs治疗过程中HBV其他编码区(尤其是免疫原性较强的C区和S区)的动态变化的监测对于认识NUCs耐药相关的肝炎突发的机制具有重要意义。同时,由于HBV具有准种特性,宿主免疫和抗病毒药物的作用可能使得该群体中出现对选择压力具有生存优势的变异株,因此阐明HBV准种的变异和进化模式对于CHB的治疗和管理具有指导价值。以往有许多的针对NUCs治疗患者HBV RT区进行的准种方面的研究。然而这些研究均只关注了NUCs作用的靶区域即RT区,而没有对HBV全长进行研究。而新近的对HBV全基因组序列的研究均采用的是PCR直接测序法扩增HBV全长序列,或通过几个扩增片段拼接得到全长序列,而没有采用克隆的方法。因此,NUCs干预下HBV准种全长序列变异及进化模式目前仍未明确。
为探讨上述问题,本研究收集了212例LAM耐药而后加用ADV治疗的患者,对不同基因型患者LAM耐药变异模式的差异以及ADV+LAM联合疗法对于不同类型LAM耐药人群的疗效进行了分析。在大样本(1 033例)的接受核苷类似物治疗的患者,采用PCR-直接测序法得到HBV RT区基因序列,进行RT区基因系统进化树构建,利用基于最大似然法的密码子替换模型分别在基因B型及C型序列中检测其可能的正选择作用位点以及差异性位点,以期从整体进化的角度分析RT区基因的进化特征。同时为探索NUCs干预下HBV准种全长序列变异及进化模式,我们纳入了5例核苷类似物序贯/联合治疗的患者以及2例未经治疗但有肝炎发作的患者,采用PCR-克隆测序-种系进化分析方法,对随访过程中的系列血清进行了HBV准种全长序列变异及进化规律的分析。
主要结果:
一、基因C型患者发生LAM突破的时限长于B型患者(P=0.036)。LAM+ADV联合治疗3个月后,所有患者平均HBV DNA水平较LAM突破时显著下降(P<0.001)。180例(84.9%)患者ALT复常,15例(11.6%)患者出现HBeAg阴转。基因B、C型患者联合治疗后HBV DNA水平、ALT复常率、HBeAg阴转率无明显差异。
二、212例患者中共计193例检测出rt204位点变异。L180M与M204V联合变异的频率显著高于M204I(76/80 vs. 28/113,P<0.001)。C型患者检出M204I+L180M变异的频率高于B型患者(P=0.002),而M204V/I+V207L变异则在B型患者中比例更高(P=0.005)。M204V+L180M、M204V/I+229及M204V/I+S213T变异在基因B、C型患者中的分布无明显差异。42例(19.8%)患者出现多位点(3个及以上位点)耐药变异。
三、M204V/I变异联合及未联合rt180、rt229、rt213位点变异组在LAM治疗基线、LAM突破及联合治疗后的HBV DNA水平、ALT水平及复常率上均无明显差异。M204V/I未合并V207L变异组联合治疗后ALT复常率高于M204V/I合并V207L变异组(P=0.001),而HBV DNA水平无明显差异。
四、基因B型RT区序列平均非同义(dN)/同义突变率(dS)比值ω=0.26,正选择位点为rt204位及rt222位氨基酸。基因C型RT区序列平均ω=0.19,正选择位点为rt180位及rt204位氨基酸。
五、基因B型及C型RT区序列有六个位点存在选择压力的差异,分别为:rt182 (P=0.08)、rt209(P=0.09)、rt211(P<0.001)、rt226(P=0.04)、rt235(P=0.08)以及rt238(P=0.02)。
六、3例LAM初治耐药而后换用其他NUCs治疗的患者HBV全基因组核苷酸及各编码区氨基酸异质性在出现LAM病毒学突破时均降至最低点,而在拯救治疗后又重新增高。接受ADV-替比夫定(LDT)序贯治疗的两例患者基因组异质性的变化则各有其特点。
七、2例未治疗组患者在出现肝炎突发时核苷酸及氨基酸异质性均较无症状期升高。
八、HBV基因组各编码区在慢性化感染过程中的遗传异质性的变化趋势各不相同,不同的患者也各有其特征。
九、5例经NUCs治疗的患者均检测出NUC相关耐药位点变异,3例患者出现多药(重)耐药。所有患者治疗前“a”决定簇均未检测出氨基酸改变,而在治疗后4例患者在该区出现了编码氨基酸的改变。未治疗的两例患者大多数的变异均集中位于前C/C区。
十、5例治疗组患者在前S/S区或C区的T细胞或B细胞表位中均出现了伴随RT区变异的编码氨基酸的改变。部分患者在核心启动子区及前C区等也检测到伴随RT区变异的核苷酸突变。
通过对这些结果进行分析并和既往研究结果进行比较,结论如下:
一、LAM+ADV联合疗法对于不同基因型(B型及C型)及不同LAM变异类型的耐药患者均能够取得较好的疗效。
二、基因B型及C型患者LAM补偿耐药位点(L180M及V207L)的分布有一定的差异。LAM初始单药治疗的患者中有一部分可能出现多位点耐药变异,提示对这部分患者初始抗病毒治疗应选择高基因屏障的药物。
三、通过大样本HBV RT区基因序列的进化分析,证实在NUCs干预下RT区基因整体上以净化选择的模式为主,少数位点则呈现为正选择的进化模式。基因B型及C型RT区基因在部分非NUC耐药变异位点上所受到的选择压力有所不同。
四、不同NUC药物治疗方式对HBV全基因组准种的进化方式有影响:采用LAM初治患者在LAM耐药时病毒异质性降低,而在挽救治疗之后异质性又逐渐升高;而ADV突破后换用其他NUC治疗的患者HBV准种则具有不同的进化规律。
五、经NUCs治疗的患者与未治疗但出现肝炎突发的患者HBV准种具有不同的进化及变异模式:未治疗患者在肝炎突发时HBV基因组变化主要集中在前C/C区,而在治疗组患者则未观察到此规律。提示引起NUCs治疗患者与未治疗患者肝炎突发的病毒学机制可能有所不同。
六、经NUCs治疗的患者在出现NUCs耐药时均检测出前S/S区和/或C区表位编码氨基酸的改变,提示HBV RT区变异可伴随有其他编码区表位的变化,使得HBV免疫原性发生改变。
Chronic hepatitis B virus (HBV) infection is a major public health problem worldwide. In recent years, treatment of chronic HBV has been improved, with the availability of nucleoside/nucleotide analogues (NUCs) such as lamivudine (LAM). Unfortunately, the clinical benefit is rarely sustained under long-term treatment due to the selection of lamivudine resistant mutants, which occur at an annual rate of 14-32%. Adefovir (ADV) has an antiviral profile with potent activity against lamivudine-resistant strains, making it useful as a rescue treatment for lamivudine resistant patients. However, suboptimal responses have also been reported after adefovir rescue therapy, and its efficacy for patients with different genotypes and different LAM-resistant mutations remains unclear.
Most studies of HBV drug resistance have focused on analysis of the A to D domain of the HBV DNA polymerase gene after detection of antiviral-resistant HBV mutants, and the drug-resistant mutational pattern that occurs in this region of the gene has been well characterized. The full HBV genome contains four partially overlapping open reading frames (ORFs) and mutations in the reverse transcriptase (RT) domain can affect the amino acid sequence of the surface protein. Previous studies have found that the immune-escape variants probably appear after antiviral-resistant variants emerge; this might be responsible for the exacerbation of chronic hepatitis B in some patients. Based on these findings, dynamic changes within other regions (especially in the immune-targeted surface and core antigen) might also be informative, and comparison of the full genome is necessary.
Like other RNA viruses, HBV shows quasispecies distribution in infected patients. Thus, to permit chronic infection management, the evolution and distribution of HBV should be monitored. These studies about the dynamic evolution of HBV quasispecies during treatment of patients with commonly used NUCs focused on the HBV DNA polymerase gene and did not investigate the mutational pattern of the full HBV genome. Several more recent studies have reported the mutational pattern of the full HBV genome. However, the HBV full genome sequence was examined by direct sequencing, not by cloning. Thus, the evolutionary pattern of complete HBV quasispecies remains unclear.
The present study investigated patterns of lamivudine-resistant mutations in 212 patients with lamivudine-resistance and evaluated the effect of these mutations on the antiviral response to LAM+ADV combination therapy. 1 033 sequences of HBV RT region derived from the patients with NUCs therapy were used to elucidate the evolution pattern and positive selection in HBV population. Meanwhile, we used full genome sequences generated from patient sera to assess the quasispecies evolutionary dynamics of five chronic hepatitis B patients who underwent sequential NUC therapies, as well as of two untreated patients with acute flares.
Main Results
1. The duration for LAM resistance in patients with genotype C was significantly longer than those with genotype B (P=0.036). Mean HBV DNA level after 3 months of LAM+ADV combination therapy was lower than that in LAM resistance (P<0.001). 180 patients (84.9%) achieved ALT normalization and 15 patients (11.6%) achieved HBeAg seroconversion. There was no difference in HBV DNA level, ALT normalization and HBeAg seroconversion rate between patients with genotype B and C.
2. rt204 mutation was detected in 193 patients. L180M was more often found in patients with M204V than those with M204I (76/80 vs. 28/113,P<0.001). M204I+L180M combined mutation presented more in patients with genotype C (P=0.002), and M204V/I+V207L was detected more in genotype B (P=0.005). There was no difference in the incidence of M204V+L180M, M204V/I+229 and M204V/I+S213T mutation between patients with genotype B and C. Multi-mutations were detected in 42 patients (19.8%).
3. The presence of the rt180、rt229、rt213 did not significantly affect viral load reduction and ALT normalization during combination therapy. The proportion of ALT normalization for patients carrying M204V/I combined with V207L was lower than those without V207L (P=0.001).
4. Mean nonsynonymous (dN)/synonymous (dS) ratioωfor genotype B sequences was 0.26; rt204 and rt222 were identified as positively selected sites. Meanωfor genotype C sequences was 0.19; rt204 and rt180 were identified as positively selected sites.
5. Significantly different selection was identified in six codons between genotype B and C sequences, including rt182 (P=0.08), rt209 (P=0.09), rt211 (P<0.001), rt226 (P=0.04), rt235 (P=0.08) and rt238 (P=0.02).
6. In the three treated patients who received lamivudine as initial antiviral therapy, nucleotide polymorphism and nonsynonymous divergence decreased at lamivudine breakthrough but increased after rescue therapies. Conversely, two other treated patients showed distinct change in divergence during adefovir-telbivudine sequential therapies.
7. Untreated subjects exhibited increased polymorphism and divergence in the preC/C region at hepatitis flare.
8. Divergence of different HBV coding regions showed different changing trend in a single host and within different patients.
9. All treated patients showed NUCs resistant mutations, three of which showed multi-drug mutations. Four of the treated patients presented amino acid changes in the“a”determinant during NUC therapy. In untreated patients, most of the variations were located in the preC/C gene.
10. All of the treated subjects showed amino acid changes within the known T-cell or B-cell epitopes in the surface or core antigen, most of which were accompanied by mutations in the RT region. Co-variations in the core promoter, the preC region and the known epitopes of the preS gene, accompanied by RT mutations, were not rare.
From these results, we draw conclusions as follows:
1. LAM and ADV combination therapy is effective for patients with genotype B and C and for patients with different LAM-resistant patterns.
2. There are differences for the LAM-resistant patterns between patients with different genotypes. Patients who receive LAM as initial therapy have risk to develop multi-mutations.
3. Purified evolution prevail HBV RT region for patients who receive NUCs therapy, with a small portion of codons under positive selection. There is difference in selection pressure between patients with genotype B and C.
4. The distribution of genetic variability of HBV shows remarkably different patterns between NUC-treated and untreated subjects and within patients receiving different NUCs therapies.
5. The underlying mechanisms of hepatitis flare differ between NUC-treated and untreated patients.
6. NUC-resistant mutations can cause amino acid changes within epitopes of other HBV regions and that the characteristics of HBV immunogenicity may change when NUC resistance emerges.
引文
1. Lee WM. Hepatitis B virus infection. N Engl J Med, 1997; 337:1733-1745.
2. Choisy M, Woelk CH, Guegan JF, et al. Comparative study of adaptive molecular evolution in different human immunodeficiency virus groups and subtypes. J Virol, 2004; 78:1962-1970.
3. Sheridan I, Pybus OG, Holmes EC, et al. High-resolution phylogenetic analysis of hepatitis C virus adaptation and its relationship to disease progression. J Virol, 2004; 78:3447-3454.
4. Brown RJ, Juttla VS, Tarr AW, et al. Evolutionary dynamics of hepatitis C virus envelope genes during chronic infection. J Gen Virol, 2005; 86:1931-1942.
5. Horiike N, Duong TN, Michitaka K, et al. Characteristics of lamivudine-resistant hepatitis B virus (HBV) strains with and without breakthrough hepatitis in patients with chronic hepatitis B evaluated by serial HBV full-genome sequences. J Med Virol, 2007; 79:911-918.
6. Enomoto M, Tamori A, Kohmoto MT, et al. Mutational patterns of hepatitis B virus genome and clinical outcomes after emergence of drug-resistant variants during lamivudine therapy: analyses of the polymerase gene and full-length sequences. J Med Virol, 2007; 79:1664-1670.
7. Conjeevaram HS, Lok AS. Management of chronic hepatitis B. J Hepatol, 2003; 38 Suppl 1:S90-103.
8. Lai CL, Dienstag J, Schiff E, et al. Prevalence and clinical correlates of YMDD variants during lamivudine therapy for patients with chronic hepatitis B. Clin Infect Dis, 2003; 36:687-696.
9. Lok AS, Lai CL, Leung N, et al. Long-term safety of lamivudine treatment in patients with chronic hepatitis B. Gastroenterology, 2003; 125:1714-1722.
10. Benhamou Y, Bochet M, Thibault V, et al. Safety and efficacy of adefovir dipivoxil in patients co-infected with HIV-1 and lamivudine-resistant hepatitis B virus: an open-label pilot study. Lancet, 2001; 358:718-723.
11. Liaw YF, Chien RN, Yeh CT. No benefit to continue lamivudine therapy afteremergence of YMDD mutations. Antivir Ther, 2004; 9:257-262.
12. Cha CK, Kwon HC, Cheong JY, et al. Association of lamivudine-resistant mutational patterns with the antiviral effect of adefovir in patients with chronic hepatitis B. J Med Virol, 2009; 81:417-424.
13. Wong VW, Chan HL, Wong ML, et al. Clinical course after stopping lamivudine in chronic hepatitis B patients with lamivudine-resistant mutants. Aliment Pharmacol Ther, 2004; 19:323-329.
14. Fung SK, Chae HB, Fontana RJ, et al. Virologic response and resistance to adefovir in patients with chronic hepatitis B. J Hepatol, 2006; 44:283-290.
15. Lok AS, McMahon BJ. Chronic hepatitis B: update of recommendations. Hepatology, 2004; 39:857-861.
16. Liaw YF. Rescue therapy for lamivudine-resistant chronic hepatitis B: when and how? Hepatology, 2007; 45:266-268.
17. Lok AS, McMahon BJ. Chronic hepatitis B. Hepatology, 2007; 45:507-539.
18. Westland CE, Yang H, Delaney WE, et al. Activity of adefovir dipivoxil against all patterns of lamivudine-resistant hepatitis B viruses in patients. J Viral Hepat, 2005; 12:67-73.
19. Suzuki F, Kumada H, Nakamura H. Changes in viral loads of lamivudine-resistant mutants and evolution of HBV sequences during adefovir dipivoxil therapy. J Med Virol, 2006; 78:1025-1034.
20. Delaney WE, Yang H, Westland CE, et al. The hepatitis B virus polymerase mutation rtV173L is selected during lamivudine therapy and enhances viral replication in vitro. J Virol, 2003; 77:11833-11841.
21. Lada O, Benhamou Y, Cahour A, et al. In vitro susceptibility of lamivudine-resistant hepatitis B virus to adefovir and tenofovir. Antivir Ther, 2004; 9:353-363.
22. Zehender G, De MC, Giambelli C, et al. Different evolutionary rates and epidemic growth of hepatitis B virus genotypes A and D. Virology, 2008; 380:84-90.
23. EASL Clinical Practice Guidelines: management of chronic hepatitis B. J Hepatol, 2009; 50:227-242.
24. Pond SL, Frost SD, Muse SV. HyPhy: hypothesis testing using phylogenies. Bioinformatics, 2005; 21:676-679.
25. Suzuki Y, Gojobori T. A method for detecting positive selection at single amino acid sites. Mol Biol Evol, 1999; 16:1315-1328.
26. Anisimova M, Bielawski JP, Yang Z. Accuracy and power of bayes prediction of amino acid sites under positive selection. Mol Biol Evol, 2002; 19:950-958.
27. Yang W, Bielawski JP, Yang Z. Widespread adaptive evolution in the human immunodeficiency virus type 1 genome. J Mol Evol, 2003; 57:212-221.
28. Nei M. Selectionism and neutralism in molecular evolution. Mol Biol Evol, 2005; 22:2318-2342.
29. Yang Z, Nielsen R, Goldman N, et al. Codon-substitution models for heterogeneous selection pressure at amino acid sites. Genetics, 2000; 155:431-449.
30. Yang Z, Nielsen R. Codon-substitution models for detecting molecular adaptation at individual sites along specific lineages. Mol Biol Evol, 2002; 19:908-917.
31. Yang Z, Swanson WJ, Vacquier VD. Maximum-likelihood analysis of molecular adaptation in abalone sperm lysin reveals variable selective pressures among lineages and sites. Mol Biol Evol, 2000; 17:1446-1455.
32. Frost SD, Nijhuis M, Schuurman R, et al. Evolution of lamivudine resistance in human immunodeficiency virus type 1-infected individuals: the relative roles of drift and selection. J Virol, 2000; 74:6262-6268.
33. Moore CB, John M, James IR, et al. Evidence of HIV-1 adaptation to HLA-restricted immune responses at a population level. Science, 2002; 296:1439-1443.
34. Harr B, Kauer M, Schlotterer C. Hitchhiking mapping: a population-based fine-mapping strategy for adaptive mutations in Drosophilamelanogaster. Proc Natl Acad Sci U S A, 2002; 99:12949-12954.
35. Tai PC, Banik D, Lin GI, et al. Novel and frequent mutations of hepatitis B virus coincide with a major histocompatibility complex class I-restricted T-cell epitope of the surface antigen. J Virol, 1997; 71:4852-4856.
36. Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis. Annu Rev Immunol, 1995; 13:29-60.
37. Liu CJ, Chen PJ, Lai MY, et al. A prospective study characterizing full-length hepatitis B virus genomes during acute exacerbation. Gastroenterology, 2003; 124:80-90.
38. Ehata T, Omata M, Chuang WL, et al. Mutations in core nucleotide sequence of hepatitis B virus correlate with fulminant and severe hepatitis. J Clin Invest, 1993; 91:1206-1213.
39. Nowak MA, Bonhoeffer S, Hill AM, et al. Viral dynamics in hepatitis B virus infection. Proc Natl Acad Sci U S A, 1996; 93:4398-4402.
40. Pawlotsky JM. The concept of hepatitis B virus mutant escape. J Clin Virol, 2005; 34 Suppl 1:S125-S129.
41. Pallier C, Castera L, Soulier A, et al. Dynamics of hepatitis B virus resistance to lamivudine. J Virol, 2006; 80:643-653.
42. Feng B, Wei L, Chen M, et al. Dynamic changes of hepatitis B virus polymerase gene including YMDD motif in lamivudine-treated patients with chronic hepatitis B. Microbiol Res, 2008; 163:487-492.
43. Ji F, Zhou L, Ma S, et al. Dynamic changes of HBV quasispecies and deletion patterns in a chronic hepatitis B patient. J Med Virol, 2009; 81:1551-1559.
44. Villet S, Ollivet A, Pichoud C, et al. Stepwise process for the development of entecavir resistance in a chronic hepatitis B virus infected patient. J Hepatol, 2007; 46:531-538.
45. Yim HJ, Hussain M, Liu Y, et al. Evolution of multi-drug resistant hepatitis B virus during sequential therapy. Hepatology, 2006; 44:703-712.
46. Guo JJ, Li QL, Shi XF, et al. Dynamics of hepatitis B virus resistance to entecavir in a nucleoside/nucleotide-naive patient. Antiviral Res, 2009; 81:180-183.
47. Chen CH, Lee CM, Tung WC, et al. Evolution of full-length HBV sequences in chronic hepatitis B patients with sequential lamivudine and adefovir dipivoxil resistance. J Hepatol, 2010; 52:478-485.
48. Inoue H, Nojima H, Okayama H. High efficiency transformation of Escherichia coli with plasmids. Gene, 1990; 96:23-28.
49. Gunther S, Li BC, Miska S, et al. A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients. J Virol, 1995; 69:5437-5444.
50. Kim H, Jee YM, Song BC, et al. Analysis of hepatitis B virus quasispecies distribution in a Korean chronic patient based on the full genome sequences. J Med Virol, 2007; 79:212-219.
51. Thompson JD, Gibson TJ, Plewniak F, et al. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res, 1997; 25:4876-4882.
52. Lole KS, Bollinger RC, Paranjape RS, et al. Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J Virol, 1999; 73:152-160.
53. Drummond AJ, Rambaut A. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol Biol, 2007; 7:214
54. Tamura K, Dudley J, Nei M, et al. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol, 2007; 24:1596-1599.
55. Librado P, Rozas J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 2009; 25:1451-1452.
56. Keulen W, van WA, Schuurman R, et al. Increased polymerase fidelity of lamivudine-resistant HIV-1 variants does not limit their evolutionary potential. AIDS, 1999; 13:1343-1349.
57. Domingo E, Gomez J. Quasispecies and its impact on viral hepatitis. Virus Res, 2007; 127:131-150.
58. Villeneuve JP, Durantel D, Durantel S, et al. Selection of a hepatitis B virus strain resistant to adefovir in a liver transplantation patient. J Hepatol, 2003; 39:1085-1089.
59. Angus P, Vaughan R, Xiong S, et al. Resistance to adefovir dipivoxil therapy associated with the selection of a novel mutation in the HBV polymerase. Gastroenterology, 2003; 125:292-297.
60. Zhou Y, Holmes EC. Bayesian estimates of the evolutionary rate and age of hepatitis B virus. J Mol Evol, 2007; 65:197-205.
61. Pfeiffer JK, Kirkegaard K. Increased fidelity reduces poliovirus fitness and virulence under selective pressure in mice. PLoS Pathog, 2005; 1:e11
62. Vignuzzi M, Stone JK, Arnold JJ, et al. Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population. Nature, 2006; 439:344-348.
63. Moreno IM, Malpica JM, Rodriguez-Cerezo E, et al. A mutation in tomato aspermy cucumovirus that abolishes cell-to-cell movement is maintained to high levels in the viral RNA population by complementation. J Virol, 1997; 71:9157-9162.
64. Brunelle MN, Jacquard AC, Pichoud C, et al. Susceptibility to antivirals of a human HBV strain with mutations conferring resistance to both lamivudine and adefovir. Hepatology, 2005; 41:1391-1398.
65. Torresi J, Earnest-Silveira L, Deliyannis G, et al. Reduced antigenicity of the hepatitis B virus HBsAg protein arising as a consequence of sequence changes in the overlapping polymerase gene that are selected by lamivudine therapy. Virology, 2002; 293:305-313.
66. Ferrari C, Penna A, Bertoletti A, et al. Cellular immune response to hepatitis B virus-encoded antigens in acute and chronic hepatitis B virus infection. J Immunol, 1990; 145:3442-3449.
67. Tsai SL, Chen PJ, Lai MY, et al. Acute exacerbations of chronic type B hepatitis are accompanied by increased T cell responses to hepatitis B core and e antigens. Implications for hepatitis B e antigen seroconversion. J Clin Invest, 1992; 89:87-96.
68. Salfeld J, Pfaff E, Noah M, et al. Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus. J Virol, 1989; 63:798-808.
69. Chuang WL, Omata M, Ehata T, et al. Precore mutations and core clustering mutations in chronic hepatitis B virus infection. Gastroenterology, 1993; 104:263-271.
70. Ferrari C, Cavalli A, Penna A, et al. Fine specificity of the human T-cell response to the hepatitis B virus preS1 antigen. Gastroenterology, 1992; 103:255-263.
71. Hong HJ, Ryu CJ, Hur H, et al. In vivo neutralization of hepatitis B virus infection by an anti-preS1 humanized antibody in chimpanzees. Virology, 2004; 318:134-141.
72. Park JH, Cho EW, Lee YJ, et al. Determination of the protective effects of neutralizing anti-hepatitis B virus (HBV) immunoglobulins by epitope mapping with recombinant HBV surface-antigen proteins. Microbiol Immunol, 2000; 44:703-710.
73. Maeng CY, Ryu CJ, Gripon P, et al. Fine mapping of virus-neutralizing epitopes on hepatitis B virus PreS1. Virology, 2000; 270:9-16.
74. Kuroki K, Floreani M, Mimms LT, et al. Epitope mapping of the PreS1 domain of the hepatitis B virus large surface protein. Virology, 1990; 176:620-624.
75. Bruss V, Thomssen R. Mapping a region of the large envelope protein required for hepatitis B virion maturation. J Virol, 1994; 68:1643-1650.
76. Blanchet M, Sureau C. Infectivity determinants of the hepatitis B virus pre-S domain are confined to the N-terminal 75 amino acid residues. J Virol, 2007; 81:5841-5849.
77. Chen CH, Changchien CS, Lee CM, et al. Combined mutations in pre-s/surface and core promoter/precore regions of hepatitis B virus increase the risk of hepatocellular carcinoma: a case-control study. J Infect Dis, 2008; 198:1634-1642.
1. Ferrer-Orta C, Arias A, Escarmis C, et al. A comparison of viral RNA-dependent RNA polymerases. Curr Opin Struct Biol, 2006; 16:27-34.
2. Batschelet E, Domingo E, Weissmann C. The proportion of revertant and mutant phage in a growing population, as a function of mutation and growth rate. Gene, 1976; 1:27-32.
3. Arnold JJ, Vignuzzi M, Stone JK, et al. Remote site control of an active site fidelity checkpoint in a viral RNA-dependent RNA polymerase. J Biol Chem, 2005; 280:25706-25716.
4. Drake JW, Holland JJ. Mutation rates among RNA viruses. Proc Natl Acad Sci U S A, 1999; 96:13910-13913.
5. Vignuzzi M, Stone JK, Arnold JJ, et al. Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population. Nature, 2006; 439:344-348.
6. Arnold JJ, Ghosh SK, Cameron CE. Poliovirus RNA-dependent RNA polymerase (3D(pol)). Divalent cation modulation of primer, template, and nucleotide selection. J Biol Chem, 1999; 274:37060-37069.
7. Domingo E, Gomez J. Quasispecies and its impact on viral hepatitis. Virus Res, 2007; 127:131-150.
8. Lopez-Bueno A, Villarreal LP, Almendral JM. Parvovirus variation for disease: a difference with RNA viruses? Curr Top Microbiol Immunol, 2006; 299:349-370.
9. Isnard M, Granier M, Frutos R, et al. Quasispecies nature of three maize streak virus isolates obtained through different modes of selection from a population used to assess response to infection of maize cultivars. J Gen Virol, 1998; 79 (Pt 12):3091-3099.
10. Sheehy AM, Gaddis NC, Choi JD, et al. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature, 2002; 418:646-650.
11. Valente L, Nishikura K. ADAR gene family and A-to-I RNA editing: diverse roles in posttranscriptional gene regulation. Prog Nucleic Acid Res Mol Biol, 2005; 79:299-338.
12. Chiu YL, Greene WC. Multifaceted antiviral actions of APOBEC3 cytidine deaminases. Trends Immunol, 2006; 27:291-297.
13. Schaub M, Keller W. RNA editing by adenosine deaminases generates RNA and protein diversity. Biochimie, 2002; 84:791-803.
14. Bonvin M, Achermann F, Greeve I, et al. Interferon-inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication. Hepatology, 2006; 43:1364-1374.
15. Suspene R, Guetard D, Henry M, et al. Extensive editing of both hepatitis B virus DNA strands by APOBEC3 cytidine deaminases in vitro and in vivo. Proc Natl Acad Sci U S A, 2005; 102:8321-8326.
16. Noguchi C, Ishino H, Tsuge M, et al. G to A hypermutation of hepatitis B virus. Hepatology, 2005; 41:626-633.
17. Gallei A, Pankraz A, Thiel HJ, et al. RNA recombination in vivo in the absence of viral replication. J Virol, 2004; 78:6271-6281.
18. Pfeiffer JK, Kirkegaard K. Increased fidelity reduces poliovirus fitness and virulence under selective pressure in mice. PLoS Pathog, 2005; 1:e11
19. Keulen W, van WA, Schuurman R, et al. Increased polymerase fidelity of lamivudine-resistant HIV-1 variants does not limit their evolutionary potential. AIDS, 1999; 13:1343-1349.
20. Moreno IM, Malpica JM, Rodriguez-Cerezo E, et al. A mutation in tomato aspermy cucumovirus that abolishes cell-to-cell movement is maintained to high levels in the viral RNA population by complementation. J Virol, 1997; 71:9157-9162.
21. Sanchez G, Bosch A, Pinto RM. Genome variability and capsid structural constraints of hepatitis a virus. J Virol, 2003; 77:452-459.
22. Eigen M. New concepts for dealing with the evolution of nucleic acids. Cold Spring Harb Symp Quant Biol, 1987; 52:307-320.
23. Saakian DB, Hu CK. Exact solution of the Eigen model with general fitness functions and degradation rates. Proc Natl Acad Sci U S A, 2006; 103:4935-4939.
24. Wilke CO. Quasispecies theory in the context of population genetics. BMC Evol Biol, 2005; 5:44
25. Eigen M. Error catastrophe and antiviral strategy. Proc Natl Acad Sci U S A, 2002; 99:13374-13376.
26. Swetina J, Schuster P. Self-replication with errors. A model for polynucleotide replication. Biophys Chem, 1982; 16:329-345.
27. de la Torre JC, Holland JJ. RNA virus quasispecies populations can suppress vastly superior mutant progeny. J Virol, 1990; 64:6278-6281.
28. Gonzalez-Lopez C, Arias A, Pariente N, et al. Preextinction viral RNA can interfere with infectivity. J Virol, 2004; 78:3319-3324.
29. Grande-Perez A, Lazaro E, Lowenstein P, et al. Suppression of viral infectivity through lethal defection. Proc Natl Acad Sci U S A, 2005; 102:4448-4452.
30. Herrera M, Garcia-Arriaza J, Pariente N, et al. Molecular basis for a lack of correlation between viral fitness and cell killing capacity. PLoS Pathog, 2007; 3:e53
31. Carrasco P, de l, I, Elena SF. Distribution of fitness and virulence effects caused by single-nucleotide substitutions in Tobacco Etch virus. J Virol, 2007; 81:12979-12984.
32. Ojosnegros S, Beerenwinkel N, Antal T, et al. Competition-colonization dynamics in an RNA virus. Proc Natl Acad Sci U S A, 2010; 107:2108-2112.
33. Delgado-Eckert E, Ojosnegros S, Beerenwinkel N. The Evolution of Virulence in RNA Viruses under a Competition-Colonization Trade-Off. Bull Math Biol, 2010;
34. Sanchez G, Bosch A, Gomez-Mariano G, et al. Evidence for quasispecies distributions in the human hepatitis A virus genome. Virology, 2003; 315:34-42.
35. Martell M, Esteban JI, Quer J, et al. Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution. J Virol, 1992; 66:3225-3229.
36. Hsu HY, Chang MH, Liaw SH, et al. Changes of hepatitis B surface antigen variants in carrier children before and after universal vaccination in Taiwan. Hepatology, 1999; 30:1312-1317.
37. Yamamoto K, Horikita M, Tsuda F, et al. Naturally occurring escape mutants of hepatitis B virus with various mutations in the S gene in carriers seropositive for antibody to hepatitis B surface antigen. J Virol, 1994; 68:2671-2676.
38. Lada O, Benhamou Y, Poynard T, et al. Coexistence of hepatitis B surface antigen (HBs Ag) and anti-HBs antibodies in chronic hepatitis B virus carriers: influence of "a" determinant variants. J Virol, 2006; 80:2968-2975.
39. Tai PC, Banik D, Lin GI, et al. Novel and frequent mutations of hepatitis B virus coincide with a major histocompatibility complex class I-restricted T-cell epitope of the surface antigen. J Virol, 1997; 71:4852-4856.
40. Margeridon S, Lachaux A, Trepo C, et al. A quasi-monoclonal anti-HBs response can lead to immune escape of 'wild-type' hepatitis B virus. J Gen Virol, 2005; 86:1687-1693.
41. Cooper S, Erickson AL, Adams EJ, et al. Analysis of a successful immune response against hepatitis C virus. Immunity, 1999; 10:439-449.
42. Timm J, Lauer GM, Kavanagh DG, et al. CD8 epitope escape and reversion in acute HCV infection. J Exp Med, 2004; 200:1593-1604.
43. Bowen DG, Walker CM. The origin of quasispecies: cause or consequence of chronic hepatitis C viral infection? J Hepatol, 2005; 42:408-417.
44. Thimme R, Bukh J, Spangenberg HC, et al. Viral and immunological determinants of hepatitis C virus clearance, persistence, and disease. Proc Natl Acad Sci U S A, 2002; 99:15661-15668.
45. Quinones-Mateu ME, Arts EJ. Virus fitness: concept, quantification, and application to HIV population dynamics. Curr Top Microbiol Immunol, 2006; 299:83-140.
46. Martin V, Perales C, Davila M, et al. Viral fitness can influence the repertoire of virus variants selected by antibodies. J Mol Biol, 2006; 362:44-54.
47. Gratton S, Cheynier R, Dumaurier MJ, et al. Highly restricted spread of HIV-1 and multiply infected cells within splenic germinal centers. Proc Natl Acad Sci U S A, 2000; 97:14566-14571.
48. Bartholomeusz A, Locarnini S. Hepatitis B virus mutations associated with antiviral therapy. J Med Virol, 2006; 78 Suppl 1:S52-S55.
49. Domingo E. RNA virus evolution and the control of viral disease. Prog Drug Res, 1989; 33:93-133.
50. Bonhoeffer S, May RM, Shaw GM, et al. Virus dynamics and drug therapy. Proc Natl Acad Sci U S A, 1997; 94:6971-6976.
51. Coffin JM. HIV population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy. Science, 1995; 267:483-489.
52. Najera I, Holguin A, Quinones-Mateu ME, et al. Pol gene quasispecies of human immunodeficiency virus: mutations associated with drug resistance in virus from patients undergoing no drug therapy. J Virol, 1995; 69:23-31.
53. Eren R, Landstein D, Terkieltaub D, et al. Preclinical evaluation of two neutralizing human monoclonal antibodies against hepatitis C virus (HCV): a potential treatment to prevent HCV reinfection in liver transplant patients. J Virol, 2006; 80:2654-2664.
54. Agol VI. Molecular mechanisms of poliovirus variation and evolution. Curr Top Microbiol Immunol, 2006; 299:211-259.
55. Tracy S, Chapman NM, Drescher KM, et al. Evolution of virulence in picornaviruses. Curr Top Microbiol Immunol, 2006; 299:193-209.
56. Kimata JT, Kuller L, Anderson DB, et al. Emerging cytopathic and antigenic simian immunodeficiency virus variants influence AIDS progression. Nat Med, 1999; 5:535-541.
57. Pawlotsky JM. Hepatitis C virus population dynamics during infection. Curr Top Microbiol Immunol, 2006; 299:261-284.
58. Matsumori A, Shimada T, Chapman NM, et al. Myocarditis and heart failure associated with hepatitis C virus infection. J Card Fail, 2006; 12:293-298.
59. Marcus PI, Rodriguez LL, Sekellick MJ. Interferon induction as a quasispecies marker of vesicular stomatitis virus populations. J Virol, 1998; 72:542-549.
60. Rowe CL, Baker SC, Nathan MJ, et al. Evolution of mouse hepatitis virus: detection and characterization of spike deletion variants during persistent infection. J Virol, 1997; 71:2959-2969.
61. Farci P, Shimoda A, Coiana A, et al. The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies. Science, 2000; 288:339-344.
62. Quer J, Esteban JI, Cos J, et al. Effect of bottlenecking on evolution of the nonstructuralprotein 3 gene of hepatitis C virus during sexually transmitted acute resolving infection. J Virol, 2005; 79:15131-15141.
63. Rothman AL, Morishima C, Bonkovsky HL, et al. Associations among clinical, immunological, and viral quasispecies measurements in advanced chronic hepatitis C. Hepatology, 2005; 41:617-625.
64. Wang XH, Netski DM, Astemborski J, et al. Progression of fibrosis during chronic hepatitis C is associated with rapid virus evolution. J Virol, 2007; 81:6513-6522.
65. Cabot B, Martell M, Esteban JI, et al. Nucleotide and amino acid complexity of hepatitis C virus quasispecies in serum and liver. J Virol, 2000; 74:805-811.
66. Jang SJ, Wang LF, Radkowski M, et al. Differences between hepatitis C virus 5' untranslated region quasispecies in serum and liver. J Gen Virol, 1999; 80 ( Pt 3):711-716.
67. Maggi F, Fornai C, Vatteroni ML, et al. Differences in hepatitis C virus quasispecies composition between liver, peripheral blood mononuclear cells and plasma. J Gen Virol, 1997; 78 ( Pt 7):1521-1525.
68. Roque-Afonso AM, Ducoulombier D, Di LG, et al. Compartmentalization of hepatitis C virus genotypes between plasma and peripheral blood mononuclear cells. J Virol, 2005; 79:6349-6357.
69. Kanazawa Y, Hayashi N, Mita E, et al. Influence of viral quasispecies on effectiveness of interferon therapy in chronic hepatitis C patients. Hepatology, 1994; 20:1121-1130.
70. Koizumi K, Enomoto N, Kurosaki M, et al. Diversity of quasispecies in various disease stages of chronic hepatitis C virus infection and its significance in interferon treatment. Hepatology, 1995; 22:30-35.
71. Le GB, Squadrito G, Nalpas B, et al. Hepatitis C virus genome complexity correlates with response to interferon therapy: a study in French patients with chronic hepatitis C. Hepatology, 1997; 25:1250-1254.
72. Farci P, Strazzera R, Alter HJ, et al. Early changes in hepatitis C viral quasispecies during interferon therapy predict the therapeutic outcome. Proc Natl Acad Sci U S A, 2002; 99:3081-3086.
73. Morishima C, Polyak SJ, Ray R, et al. Hepatitis C virus-specific immune responses and quasi-species variability at baseline are associated with nonresponse to antiviral therapy during advanced hepatitis C. J Infect Dis, 2006; 193:931-940.
74. Enomoto N, Sakuma I, Asahina Y, et al. Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus 1b. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region. J Clin Invest, 1995; 96:224-230.
75. Chambers TJ, Fan X, Droll DA, et al. Quasispecies heterogeneity within the E1/E2 region as a pretreatment variable during pegylated interferon therapy of chronic hepatitis C virus infection. J Virol, 2005; 79:3071-3083.
76. Squadrito G, Leone F, Sartori M, et al. Mutations in the nonstructural 5A region of hepatitis C virus and response of chronic hepatitis C to interferon alfa. Gastroenterology, 1997; 113:567-572.
77. Chen L, Zhang Q, Yu DM, et al. Early changes of hepatitis B virus quasispecies during lamivudine treatment and the correlation with antiviral efficacy. J Hepatol, 2009; 50:895-905.
78. Liu F, Chen L, Yu DM, et al. Evolutionary patterns of hepatitis B virus quasispecies under different selective pressures: correlation with antiviral efficacy. Gut, 2011;
2. Choisy M, Woelk CH, Guegan JF, et al. Comparative study of adaptive molecular evolution in different human immunodeficiency virus groups and subtypes. J Virol, 2004; 78:1962-1970.
3. Sheridan I, Pybus OG, Holmes EC, et al. High-resolution phylogenetic analysis of hepatitis C virus adaptation and its relationship to disease progression. J Virol, 2004; 78:3447-3454.
4. Brown RJ, Juttla VS, Tarr AW, et al. Evolutionary dynamics of hepatitis C virus envelope genes during chronic infection. J Gen Virol, 2005; 86:1931-1942.
5. Horiike N, Duong TN, Michitaka K, et al. Characteristics of lamivudine-resistant hepatitis B virus (HBV) strains with and without breakthrough hepatitis in patients with chronic hepatitis B evaluated by serial HBV full-genome sequences. J Med Virol, 2007; 79:911-918.
6. Enomoto M, Tamori A, Kohmoto MT, et al. Mutational patterns of hepatitis B virus genome and clinical outcomes after emergence of drug-resistant variants during lamivudine therapy: analyses of the polymerase gene and full-length sequences. J Med Virol, 2007; 79:1664-1670.
7. Conjeevaram HS, Lok AS. Management of chronic hepatitis B. J Hepatol, 2003; 38 Suppl 1:S90-103.
8. Lai CL, Dienstag J, Schiff E, et al. Prevalence and clinical correlates of YMDD variants during lamivudine therapy for patients with chronic hepatitis B. Clin Infect Dis, 2003; 36:687-696.
9. Lok AS, Lai CL, Leung N, et al. Long-term safety of lamivudine treatment in patients with chronic hepatitis B. Gastroenterology, 2003; 125:1714-1722.
10. Benhamou Y, Bochet M, Thibault V, et al. Safety and efficacy of adefovir dipivoxil in patients co-infected with HIV-1 and lamivudine-resistant hepatitis B virus: an open-label pilot study. Lancet, 2001; 358:718-723.
11. Liaw YF, Chien RN, Yeh CT. No benefit to continue lamivudine therapy afteremergence of YMDD mutations. Antivir Ther, 2004; 9:257-262.
12. Cha CK, Kwon HC, Cheong JY, et al. Association of lamivudine-resistant mutational patterns with the antiviral effect of adefovir in patients with chronic hepatitis B. J Med Virol, 2009; 81:417-424.
13. Wong VW, Chan HL, Wong ML, et al. Clinical course after stopping lamivudine in chronic hepatitis B patients with lamivudine-resistant mutants. Aliment Pharmacol Ther, 2004; 19:323-329.
14. Fung SK, Chae HB, Fontana RJ, et al. Virologic response and resistance to adefovir in patients with chronic hepatitis B. J Hepatol, 2006; 44:283-290.
15. Lok AS, McMahon BJ. Chronic hepatitis B: update of recommendations. Hepatology, 2004; 39:857-861.
16. Liaw YF. Rescue therapy for lamivudine-resistant chronic hepatitis B: when and how? Hepatology, 2007; 45:266-268.
17. Lok AS, McMahon BJ. Chronic hepatitis B. Hepatology, 2007; 45:507-539.
18. Westland CE, Yang H, Delaney WE, et al. Activity of adefovir dipivoxil against all patterns of lamivudine-resistant hepatitis B viruses in patients. J Viral Hepat, 2005; 12:67-73.
19. Suzuki F, Kumada H, Nakamura H. Changes in viral loads of lamivudine-resistant mutants and evolution of HBV sequences during adefovir dipivoxil therapy. J Med Virol, 2006; 78:1025-1034.
20. Delaney WE, Yang H, Westland CE, et al. The hepatitis B virus polymerase mutation rtV173L is selected during lamivudine therapy and enhances viral replication in vitro. J Virol, 2003; 77:11833-11841.
21. Lada O, Benhamou Y, Cahour A, et al. In vitro susceptibility of lamivudine-resistant hepatitis B virus to adefovir and tenofovir. Antivir Ther, 2004; 9:353-363.
22. Zehender G, De MC, Giambelli C, et al. Different evolutionary rates and epidemic growth of hepatitis B virus genotypes A and D. Virology, 2008; 380:84-90.
23. EASL Clinical Practice Guidelines: management of chronic hepatitis B. J Hepatol, 2009; 50:227-242.
24. Pond SL, Frost SD, Muse SV. HyPhy: hypothesis testing using phylogenies. Bioinformatics, 2005; 21:676-679.
25. Suzuki Y, Gojobori T. A method for detecting positive selection at single amino acid sites. Mol Biol Evol, 1999; 16:1315-1328.
26. Anisimova M, Bielawski JP, Yang Z. Accuracy and power of bayes prediction of amino acid sites under positive selection. Mol Biol Evol, 2002; 19:950-958.
27. Yang W, Bielawski JP, Yang Z. Widespread adaptive evolution in the human immunodeficiency virus type 1 genome. J Mol Evol, 2003; 57:212-221.
28. Nei M. Selectionism and neutralism in molecular evolution. Mol Biol Evol, 2005; 22:2318-2342.
29. Yang Z, Nielsen R, Goldman N, et al. Codon-substitution models for heterogeneous selection pressure at amino acid sites. Genetics, 2000; 155:431-449.
30. Yang Z, Nielsen R. Codon-substitution models for detecting molecular adaptation at individual sites along specific lineages. Mol Biol Evol, 2002; 19:908-917.
31. Yang Z, Swanson WJ, Vacquier VD. Maximum-likelihood analysis of molecular adaptation in abalone sperm lysin reveals variable selective pressures among lineages and sites. Mol Biol Evol, 2000; 17:1446-1455.
32. Frost SD, Nijhuis M, Schuurman R, et al. Evolution of lamivudine resistance in human immunodeficiency virus type 1-infected individuals: the relative roles of drift and selection. J Virol, 2000; 74:6262-6268.
33. Moore CB, John M, James IR, et al. Evidence of HIV-1 adaptation to HLA-restricted immune responses at a population level. Science, 2002; 296:1439-1443.
34. Harr B, Kauer M, Schlotterer C. Hitchhiking mapping: a population-based fine-mapping strategy for adaptive mutations in Drosophilamelanogaster. Proc Natl Acad Sci U S A, 2002; 99:12949-12954.
35. Tai PC, Banik D, Lin GI, et al. Novel and frequent mutations of hepatitis B virus coincide with a major histocompatibility complex class I-restricted T-cell epitope of the surface antigen. J Virol, 1997; 71:4852-4856.
36. Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis. Annu Rev Immunol, 1995; 13:29-60.
37. Liu CJ, Chen PJ, Lai MY, et al. A prospective study characterizing full-length hepatitis B virus genomes during acute exacerbation. Gastroenterology, 2003; 124:80-90.
38. Ehata T, Omata M, Chuang WL, et al. Mutations in core nucleotide sequence of hepatitis B virus correlate with fulminant and severe hepatitis. J Clin Invest, 1993; 91:1206-1213.
39. Nowak MA, Bonhoeffer S, Hill AM, et al. Viral dynamics in hepatitis B virus infection. Proc Natl Acad Sci U S A, 1996; 93:4398-4402.
40. Pawlotsky JM. The concept of hepatitis B virus mutant escape. J Clin Virol, 2005; 34 Suppl 1:S125-S129.
41. Pallier C, Castera L, Soulier A, et al. Dynamics of hepatitis B virus resistance to lamivudine. J Virol, 2006; 80:643-653.
42. Feng B, Wei L, Chen M, et al. Dynamic changes of hepatitis B virus polymerase gene including YMDD motif in lamivudine-treated patients with chronic hepatitis B. Microbiol Res, 2008; 163:487-492.
43. Ji F, Zhou L, Ma S, et al. Dynamic changes of HBV quasispecies and deletion patterns in a chronic hepatitis B patient. J Med Virol, 2009; 81:1551-1559.
44. Villet S, Ollivet A, Pichoud C, et al. Stepwise process for the development of entecavir resistance in a chronic hepatitis B virus infected patient. J Hepatol, 2007; 46:531-538.
45. Yim HJ, Hussain M, Liu Y, et al. Evolution of multi-drug resistant hepatitis B virus during sequential therapy. Hepatology, 2006; 44:703-712.
46. Guo JJ, Li QL, Shi XF, et al. Dynamics of hepatitis B virus resistance to entecavir in a nucleoside/nucleotide-naive patient. Antiviral Res, 2009; 81:180-183.
47. Chen CH, Lee CM, Tung WC, et al. Evolution of full-length HBV sequences in chronic hepatitis B patients with sequential lamivudine and adefovir dipivoxil resistance. J Hepatol, 2010; 52:478-485.
48. Inoue H, Nojima H, Okayama H. High efficiency transformation of Escherichia coli with plasmids. Gene, 1990; 96:23-28.
49. Gunther S, Li BC, Miska S, et al. A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients. J Virol, 1995; 69:5437-5444.
50. Kim H, Jee YM, Song BC, et al. Analysis of hepatitis B virus quasispecies distribution in a Korean chronic patient based on the full genome sequences. J Med Virol, 2007; 79:212-219.
51. Thompson JD, Gibson TJ, Plewniak F, et al. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res, 1997; 25:4876-4882.
52. Lole KS, Bollinger RC, Paranjape RS, et al. Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J Virol, 1999; 73:152-160.
53. Drummond AJ, Rambaut A. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol Biol, 2007; 7:214
54. Tamura K, Dudley J, Nei M, et al. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol, 2007; 24:1596-1599.
55. Librado P, Rozas J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 2009; 25:1451-1452.
56. Keulen W, van WA, Schuurman R, et al. Increased polymerase fidelity of lamivudine-resistant HIV-1 variants does not limit their evolutionary potential. AIDS, 1999; 13:1343-1349.
57. Domingo E, Gomez J. Quasispecies and its impact on viral hepatitis. Virus Res, 2007; 127:131-150.
58. Villeneuve JP, Durantel D, Durantel S, et al. Selection of a hepatitis B virus strain resistant to adefovir in a liver transplantation patient. J Hepatol, 2003; 39:1085-1089.
59. Angus P, Vaughan R, Xiong S, et al. Resistance to adefovir dipivoxil therapy associated with the selection of a novel mutation in the HBV polymerase. Gastroenterology, 2003; 125:292-297.
60. Zhou Y, Holmes EC. Bayesian estimates of the evolutionary rate and age of hepatitis B virus. J Mol Evol, 2007; 65:197-205.
61. Pfeiffer JK, Kirkegaard K. Increased fidelity reduces poliovirus fitness and virulence under selective pressure in mice. PLoS Pathog, 2005; 1:e11
62. Vignuzzi M, Stone JK, Arnold JJ, et al. Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population. Nature, 2006; 439:344-348.
63. Moreno IM, Malpica JM, Rodriguez-Cerezo E, et al. A mutation in tomato aspermy cucumovirus that abolishes cell-to-cell movement is maintained to high levels in the viral RNA population by complementation. J Virol, 1997; 71:9157-9162.
64. Brunelle MN, Jacquard AC, Pichoud C, et al. Susceptibility to antivirals of a human HBV strain with mutations conferring resistance to both lamivudine and adefovir. Hepatology, 2005; 41:1391-1398.
65. Torresi J, Earnest-Silveira L, Deliyannis G, et al. Reduced antigenicity of the hepatitis B virus HBsAg protein arising as a consequence of sequence changes in the overlapping polymerase gene that are selected by lamivudine therapy. Virology, 2002; 293:305-313.
66. Ferrari C, Penna A, Bertoletti A, et al. Cellular immune response to hepatitis B virus-encoded antigens in acute and chronic hepatitis B virus infection. J Immunol, 1990; 145:3442-3449.
67. Tsai SL, Chen PJ, Lai MY, et al. Acute exacerbations of chronic type B hepatitis are accompanied by increased T cell responses to hepatitis B core and e antigens. Implications for hepatitis B e antigen seroconversion. J Clin Invest, 1992; 89:87-96.
68. Salfeld J, Pfaff E, Noah M, et al. Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus. J Virol, 1989; 63:798-808.
69. Chuang WL, Omata M, Ehata T, et al. Precore mutations and core clustering mutations in chronic hepatitis B virus infection. Gastroenterology, 1993; 104:263-271.
70. Ferrari C, Cavalli A, Penna A, et al. Fine specificity of the human T-cell response to the hepatitis B virus preS1 antigen. Gastroenterology, 1992; 103:255-263.
71. Hong HJ, Ryu CJ, Hur H, et al. In vivo neutralization of hepatitis B virus infection by an anti-preS1 humanized antibody in chimpanzees. Virology, 2004; 318:134-141.
72. Park JH, Cho EW, Lee YJ, et al. Determination of the protective effects of neutralizing anti-hepatitis B virus (HBV) immunoglobulins by epitope mapping with recombinant HBV surface-antigen proteins. Microbiol Immunol, 2000; 44:703-710.
73. Maeng CY, Ryu CJ, Gripon P, et al. Fine mapping of virus-neutralizing epitopes on hepatitis B virus PreS1. Virology, 2000; 270:9-16.
74. Kuroki K, Floreani M, Mimms LT, et al. Epitope mapping of the PreS1 domain of the hepatitis B virus large surface protein. Virology, 1990; 176:620-624.
75. Bruss V, Thomssen R. Mapping a region of the large envelope protein required for hepatitis B virion maturation. J Virol, 1994; 68:1643-1650.
76. Blanchet M, Sureau C. Infectivity determinants of the hepatitis B virus pre-S domain are confined to the N-terminal 75 amino acid residues. J Virol, 2007; 81:5841-5849.
77. Chen CH, Changchien CS, Lee CM, et al. Combined mutations in pre-s/surface and core promoter/precore regions of hepatitis B virus increase the risk of hepatocellular carcinoma: a case-control study. J Infect Dis, 2008; 198:1634-1642.
1. Ferrer-Orta C, Arias A, Escarmis C, et al. A comparison of viral RNA-dependent RNA polymerases. Curr Opin Struct Biol, 2006; 16:27-34.
2. Batschelet E, Domingo E, Weissmann C. The proportion of revertant and mutant phage in a growing population, as a function of mutation and growth rate. Gene, 1976; 1:27-32.
3. Arnold JJ, Vignuzzi M, Stone JK, et al. Remote site control of an active site fidelity checkpoint in a viral RNA-dependent RNA polymerase. J Biol Chem, 2005; 280:25706-25716.
4. Drake JW, Holland JJ. Mutation rates among RNA viruses. Proc Natl Acad Sci U S A, 1999; 96:13910-13913.
5. Vignuzzi M, Stone JK, Arnold JJ, et al. Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population. Nature, 2006; 439:344-348.
6. Arnold JJ, Ghosh SK, Cameron CE. Poliovirus RNA-dependent RNA polymerase (3D(pol)). Divalent cation modulation of primer, template, and nucleotide selection. J Biol Chem, 1999; 274:37060-37069.
7. Domingo E, Gomez J. Quasispecies and its impact on viral hepatitis. Virus Res, 2007; 127:131-150.
8. Lopez-Bueno A, Villarreal LP, Almendral JM. Parvovirus variation for disease: a difference with RNA viruses? Curr Top Microbiol Immunol, 2006; 299:349-370.
9. Isnard M, Granier M, Frutos R, et al. Quasispecies nature of three maize streak virus isolates obtained through different modes of selection from a population used to assess response to infection of maize cultivars. J Gen Virol, 1998; 79 (Pt 12):3091-3099.
10. Sheehy AM, Gaddis NC, Choi JD, et al. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature, 2002; 418:646-650.
11. Valente L, Nishikura K. ADAR gene family and A-to-I RNA editing: diverse roles in posttranscriptional gene regulation. Prog Nucleic Acid Res Mol Biol, 2005; 79:299-338.
12. Chiu YL, Greene WC. Multifaceted antiviral actions of APOBEC3 cytidine deaminases. Trends Immunol, 2006; 27:291-297.
13. Schaub M, Keller W. RNA editing by adenosine deaminases generates RNA and protein diversity. Biochimie, 2002; 84:791-803.
14. Bonvin M, Achermann F, Greeve I, et al. Interferon-inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication. Hepatology, 2006; 43:1364-1374.
15. Suspene R, Guetard D, Henry M, et al. Extensive editing of both hepatitis B virus DNA strands by APOBEC3 cytidine deaminases in vitro and in vivo. Proc Natl Acad Sci U S A, 2005; 102:8321-8326.
16. Noguchi C, Ishino H, Tsuge M, et al. G to A hypermutation of hepatitis B virus. Hepatology, 2005; 41:626-633.
17. Gallei A, Pankraz A, Thiel HJ, et al. RNA recombination in vivo in the absence of viral replication. J Virol, 2004; 78:6271-6281.
18. Pfeiffer JK, Kirkegaard K. Increased fidelity reduces poliovirus fitness and virulence under selective pressure in mice. PLoS Pathog, 2005; 1:e11
19. Keulen W, van WA, Schuurman R, et al. Increased polymerase fidelity of lamivudine-resistant HIV-1 variants does not limit their evolutionary potential. AIDS, 1999; 13:1343-1349.
20. Moreno IM, Malpica JM, Rodriguez-Cerezo E, et al. A mutation in tomato aspermy cucumovirus that abolishes cell-to-cell movement is maintained to high levels in the viral RNA population by complementation. J Virol, 1997; 71:9157-9162.
21. Sanchez G, Bosch A, Pinto RM. Genome variability and capsid structural constraints of hepatitis a virus. J Virol, 2003; 77:452-459.
22. Eigen M. New concepts for dealing with the evolution of nucleic acids. Cold Spring Harb Symp Quant Biol, 1987; 52:307-320.
23. Saakian DB, Hu CK. Exact solution of the Eigen model with general fitness functions and degradation rates. Proc Natl Acad Sci U S A, 2006; 103:4935-4939.
24. Wilke CO. Quasispecies theory in the context of population genetics. BMC Evol Biol, 2005; 5:44
25. Eigen M. Error catastrophe and antiviral strategy. Proc Natl Acad Sci U S A, 2002; 99:13374-13376.
26. Swetina J, Schuster P. Self-replication with errors. A model for polynucleotide replication. Biophys Chem, 1982; 16:329-345.
27. de la Torre JC, Holland JJ. RNA virus quasispecies populations can suppress vastly superior mutant progeny. J Virol, 1990; 64:6278-6281.
28. Gonzalez-Lopez C, Arias A, Pariente N, et al. Preextinction viral RNA can interfere with infectivity. J Virol, 2004; 78:3319-3324.
29. Grande-Perez A, Lazaro E, Lowenstein P, et al. Suppression of viral infectivity through lethal defection. Proc Natl Acad Sci U S A, 2005; 102:4448-4452.
30. Herrera M, Garcia-Arriaza J, Pariente N, et al. Molecular basis for a lack of correlation between viral fitness and cell killing capacity. PLoS Pathog, 2007; 3:e53
31. Carrasco P, de l, I, Elena SF. Distribution of fitness and virulence effects caused by single-nucleotide substitutions in Tobacco Etch virus. J Virol, 2007; 81:12979-12984.
32. Ojosnegros S, Beerenwinkel N, Antal T, et al. Competition-colonization dynamics in an RNA virus. Proc Natl Acad Sci U S A, 2010; 107:2108-2112.
33. Delgado-Eckert E, Ojosnegros S, Beerenwinkel N. The Evolution of Virulence in RNA Viruses under a Competition-Colonization Trade-Off. Bull Math Biol, 2010;
34. Sanchez G, Bosch A, Gomez-Mariano G, et al. Evidence for quasispecies distributions in the human hepatitis A virus genome. Virology, 2003; 315:34-42.
35. Martell M, Esteban JI, Quer J, et al. Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution. J Virol, 1992; 66:3225-3229.
36. Hsu HY, Chang MH, Liaw SH, et al. Changes of hepatitis B surface antigen variants in carrier children before and after universal vaccination in Taiwan. Hepatology, 1999; 30:1312-1317.
37. Yamamoto K, Horikita M, Tsuda F, et al. Naturally occurring escape mutants of hepatitis B virus with various mutations in the S gene in carriers seropositive for antibody to hepatitis B surface antigen. J Virol, 1994; 68:2671-2676.
38. Lada O, Benhamou Y, Poynard T, et al. Coexistence of hepatitis B surface antigen (HBs Ag) and anti-HBs antibodies in chronic hepatitis B virus carriers: influence of "a" determinant variants. J Virol, 2006; 80:2968-2975.
39. Tai PC, Banik D, Lin GI, et al. Novel and frequent mutations of hepatitis B virus coincide with a major histocompatibility complex class I-restricted T-cell epitope of the surface antigen. J Virol, 1997; 71:4852-4856.
40. Margeridon S, Lachaux A, Trepo C, et al. A quasi-monoclonal anti-HBs response can lead to immune escape of 'wild-type' hepatitis B virus. J Gen Virol, 2005; 86:1687-1693.
41. Cooper S, Erickson AL, Adams EJ, et al. Analysis of a successful immune response against hepatitis C virus. Immunity, 1999; 10:439-449.
42. Timm J, Lauer GM, Kavanagh DG, et al. CD8 epitope escape and reversion in acute HCV infection. J Exp Med, 2004; 200:1593-1604.
43. Bowen DG, Walker CM. The origin of quasispecies: cause or consequence of chronic hepatitis C viral infection? J Hepatol, 2005; 42:408-417.
44. Thimme R, Bukh J, Spangenberg HC, et al. Viral and immunological determinants of hepatitis C virus clearance, persistence, and disease. Proc Natl Acad Sci U S A, 2002; 99:15661-15668.
45. Quinones-Mateu ME, Arts EJ. Virus fitness: concept, quantification, and application to HIV population dynamics. Curr Top Microbiol Immunol, 2006; 299:83-140.
46. Martin V, Perales C, Davila M, et al. Viral fitness can influence the repertoire of virus variants selected by antibodies. J Mol Biol, 2006; 362:44-54.
47. Gratton S, Cheynier R, Dumaurier MJ, et al. Highly restricted spread of HIV-1 and multiply infected cells within splenic germinal centers. Proc Natl Acad Sci U S A, 2000; 97:14566-14571.
48. Bartholomeusz A, Locarnini S. Hepatitis B virus mutations associated with antiviral therapy. J Med Virol, 2006; 78 Suppl 1:S52-S55.
49. Domingo E. RNA virus evolution and the control of viral disease. Prog Drug Res, 1989; 33:93-133.
50. Bonhoeffer S, May RM, Shaw GM, et al. Virus dynamics and drug therapy. Proc Natl Acad Sci U S A, 1997; 94:6971-6976.
51. Coffin JM. HIV population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy. Science, 1995; 267:483-489.
52. Najera I, Holguin A, Quinones-Mateu ME, et al. Pol gene quasispecies of human immunodeficiency virus: mutations associated with drug resistance in virus from patients undergoing no drug therapy. J Virol, 1995; 69:23-31.
53. Eren R, Landstein D, Terkieltaub D, et al. Preclinical evaluation of two neutralizing human monoclonal antibodies against hepatitis C virus (HCV): a potential treatment to prevent HCV reinfection in liver transplant patients. J Virol, 2006; 80:2654-2664.
54. Agol VI. Molecular mechanisms of poliovirus variation and evolution. Curr Top Microbiol Immunol, 2006; 299:211-259.
55. Tracy S, Chapman NM, Drescher KM, et al. Evolution of virulence in picornaviruses. Curr Top Microbiol Immunol, 2006; 299:193-209.
56. Kimata JT, Kuller L, Anderson DB, et al. Emerging cytopathic and antigenic simian immunodeficiency virus variants influence AIDS progression. Nat Med, 1999; 5:535-541.
57. Pawlotsky JM. Hepatitis C virus population dynamics during infection. Curr Top Microbiol Immunol, 2006; 299:261-284.
58. Matsumori A, Shimada T, Chapman NM, et al. Myocarditis and heart failure associated with hepatitis C virus infection. J Card Fail, 2006; 12:293-298.
59. Marcus PI, Rodriguez LL, Sekellick MJ. Interferon induction as a quasispecies marker of vesicular stomatitis virus populations. J Virol, 1998; 72:542-549.
60. Rowe CL, Baker SC, Nathan MJ, et al. Evolution of mouse hepatitis virus: detection and characterization of spike deletion variants during persistent infection. J Virol, 1997; 71:2959-2969.
61. Farci P, Shimoda A, Coiana A, et al. The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies. Science, 2000; 288:339-344.
62. Quer J, Esteban JI, Cos J, et al. Effect of bottlenecking on evolution of the nonstructuralprotein 3 gene of hepatitis C virus during sexually transmitted acute resolving infection. J Virol, 2005; 79:15131-15141.
63. Rothman AL, Morishima C, Bonkovsky HL, et al. Associations among clinical, immunological, and viral quasispecies measurements in advanced chronic hepatitis C. Hepatology, 2005; 41:617-625.
64. Wang XH, Netski DM, Astemborski J, et al. Progression of fibrosis during chronic hepatitis C is associated with rapid virus evolution. J Virol, 2007; 81:6513-6522.
65. Cabot B, Martell M, Esteban JI, et al. Nucleotide and amino acid complexity of hepatitis C virus quasispecies in serum and liver. J Virol, 2000; 74:805-811.
66. Jang SJ, Wang LF, Radkowski M, et al. Differences between hepatitis C virus 5' untranslated region quasispecies in serum and liver. J Gen Virol, 1999; 80 ( Pt 3):711-716.
67. Maggi F, Fornai C, Vatteroni ML, et al. Differences in hepatitis C virus quasispecies composition between liver, peripheral blood mononuclear cells and plasma. J Gen Virol, 1997; 78 ( Pt 7):1521-1525.
68. Roque-Afonso AM, Ducoulombier D, Di LG, et al. Compartmentalization of hepatitis C virus genotypes between plasma and peripheral blood mononuclear cells. J Virol, 2005; 79:6349-6357.
69. Kanazawa Y, Hayashi N, Mita E, et al. Influence of viral quasispecies on effectiveness of interferon therapy in chronic hepatitis C patients. Hepatology, 1994; 20:1121-1130.
70. Koizumi K, Enomoto N, Kurosaki M, et al. Diversity of quasispecies in various disease stages of chronic hepatitis C virus infection and its significance in interferon treatment. Hepatology, 1995; 22:30-35.
71. Le GB, Squadrito G, Nalpas B, et al. Hepatitis C virus genome complexity correlates with response to interferon therapy: a study in French patients with chronic hepatitis C. Hepatology, 1997; 25:1250-1254.
72. Farci P, Strazzera R, Alter HJ, et al. Early changes in hepatitis C viral quasispecies during interferon therapy predict the therapeutic outcome. Proc Natl Acad Sci U S A, 2002; 99:3081-3086.
73. Morishima C, Polyak SJ, Ray R, et al. Hepatitis C virus-specific immune responses and quasi-species variability at baseline are associated with nonresponse to antiviral therapy during advanced hepatitis C. J Infect Dis, 2006; 193:931-940.
74. Enomoto N, Sakuma I, Asahina Y, et al. Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus 1b. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region. J Clin Invest, 1995; 96:224-230.
75. Chambers TJ, Fan X, Droll DA, et al. Quasispecies heterogeneity within the E1/E2 region as a pretreatment variable during pegylated interferon therapy of chronic hepatitis C virus infection. J Virol, 2005; 79:3071-3083.
76. Squadrito G, Leone F, Sartori M, et al. Mutations in the nonstructural 5A region of hepatitis C virus and response of chronic hepatitis C to interferon alfa. Gastroenterology, 1997; 113:567-572.
77. Chen L, Zhang Q, Yu DM, et al. Early changes of hepatitis B virus quasispecies during lamivudine treatment and the correlation with antiviral efficacy. J Hepatol, 2009; 50:895-905.
78. Liu F, Chen L, Yu DM, et al. Evolutionary patterns of hepatitis B virus quasispecies under different selective pressures: correlation with antiviral efficacy. Gut, 2011;