单个肝细胞核内鸭乙型肝炎病毒DNA的水平及动态变化
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摘要
研究背景、目的及意义：
共价闭合环状DNA (covalently closed circular DNA, cccDNA)是乙型肝炎病毒(Hepatitis B virus, HBV)的复制模板,长期稳定的以微染色体的形式存在于受感染的肝细胞核内,虽其含量远低于HBV存在的一般形式松弛环状DNA(relaxed circular DNA, rcDNA),但对HBV的复制和感染状态的建立具有举足轻重的意义。cccDNA是HBV感染状态的持续、停用抗病毒药物后病情反复及药物耐药的关键因素,因此只有清除或有效降低细胞核内的cccDNA,才能彻底消除乙肝患者病毒携带状态,或使病情趋于稳定。
cccDNA的定量检测对于理解HBV的复制和清除,评估抗病毒药物的疗效和疗程等具有重要的意义；但rcDNA或双链线性DNA (double strand DNA, dsDNA)的非特异性扩增仍然是目前要面临的主要问题。检测方法不成熟和肝组织来源困难在一定程度上阻碍了对乙肝患者cccDNA的直接研究。
在鸭乙型肝炎病毒(Duck hepatitis B virus, DHBV)感染的鸭肝细胞,胞核内病毒DNA主要是以cccDNA为主,而胞浆内作为cccDNA前体的rcDNA可能有其量的10倍。因此定量检测核内病毒DNA可较直接反映cccDNA的水平。
本研究将在单个肝细胞核的层面上定量检测慢性DHBV感染状态下核内病毒DNA水平及其影响因素；通过恩替卡韦短期抑制病毒复制,观察血清DHBVDNA阴转时及阴转后单个肝细胞核内病毒水平及受感染肝细胞核数的动态变化。
我们的研究首次提出了在单个肝细胞核的水平上分析核内病毒DNA,并通过恩替卡韦抑制病毒复制观察核内病毒DNA的清除动力学。完成这一研究会帮助我们更加深入理解嗜肝DNA病毒的生活周期,病毒与宿主细胞间的相互作用,药物作用下单个核内病毒DNA的清除规律,同时也将为下一步研究慢乙肝患者核内HBV DNA水平及抗病毒疗效评估等奠定基础。
研究方法：
第一部分：建立鸭乙型肝炎病毒后天感染模型
筛选1和58日龄广东樱桃谷鸭各20只,在2日及2月龄时病毒阴性动物静脉接种含1×108DHBV DNA病毒颗粒的血清。接种后不同时间点采血,抽提DNA,PCR定性及定量检测外周血病毒DNA水平。
第二部分：慢性感染状态下单个肝细胞核内DHBV DNA水平
1.对纳入的11只45日龄慢性DHBV感染鸭行肝活检手术
2.建立单个核内DHBV DNA定量检测方法
采用Taqman探针荧光定量PCR法检测单个核内DHBV DNA水平,并验证该方法的敏感性、特异性及批间差异：
已知浓度的标准质粒(PBS-DHBV1.2)倍比稀释为1、10、100、1000和2、20、200、2000copies/μl。按照50μl的反应体系分别向反应孔内加入1、10、100、1000和2、20、200、2000拷贝数的PBS-DHBV1.2质粒进行real-time PCR定量扩增,得到扩增曲线和标准曲线,确定方法的敏感性(最低检测下限)；
分别用含有DHBV、HBV及HCV全基因组的质粒进行扩增,验证引物和探针的特异性；
分四批定量检测两只动物单个核内DHBV DNA水平,计算核内病毒拷贝数的批间差异,用变异系数(CV%)表示。
3.匀浆研磨5mg肝组织、低速离心、匀浆液充分洗涤得到肝细胞核悬液、通过流式细胞仪分选得到单个细胞核；单个细胞核经蛋白酶K消化、EcoRI酶切后,采用荧光定量PCR法检测核内病毒DNA水平。
4.流式细胞仪分选105个肝细胞核至1ml的PBS溶液中、提取总的核内DNA,采用荧光定量PCR法检测总的核内病毒DNA水平。
5.细胞周期检测并分选处在不同周期的单个核
500μ1的肝细胞核悬液(约1×105个核)中加入15μl PI(100μg/ml)染色液,避光室温孵育至少30min;流式细胞仪将根据核内DNA的量确定细胞周期的分布状况,并分选处在不同细胞周期的单个核至96孔板中。
6.明确整合的病毒DNA影响
质粒安全ATP依赖的DNA酶(PSAD)可以选择性消化线性DNA(即整合的片段),对cccDNA和rcDNA无影响。单个核内病毒DNA在1个单位PSAD酶的作用下37℃孵育30min,然后70℃、30min灭活该酶;再加入5个单位的EcoR I,37℃孵育30min。采用荧光定量PCR法检测单个核内病毒DNA水平,确定病毒阳性细胞核数,比较PSAD处理组与未处理组间的差异。
7.病毒DNA在肝内的存在形式
采用酚-氯仿抽提法提取肝细胞核、细胞质及肝细胞内的总DNA, Southern blot印迹杂交检测病毒DNA的存在形式。
第三部分：恩替卡韦治疗对单个核内DHBV DNA的影响
实验分组：将11只45日龄慢性DHBV感染鸭随机分两组：恩替卡韦治疗组(n=6)和未治疗组(n=5)。
治疗组每只动物每日接受0.5mg恩替卡韦抗病毒,血清DHBV DNA阴转时,行第二次肝活检；阴转后继续给药治疗16周,治疗结束时处死所用动物,取出肝组织标本。
观察单个核内DHBV DNA水平及受感染肝细胞核数的动态变化。
统计学分析：
所有数据采用SPSS16.0进行处理。计数资料组间比较采用Pearson's chi-square检验,对理论频数小于5的四格表资料采用Fisher精确概率法。连续变量之间的相关性应用Pearson方法。计量资料若满足正态分布或方差齐性采用参数检验方法(两独立样本t检验)；计量资料若不满足正态分布及方差不齐则采用非参数检验方法(Mann-Whitney U检验)。计量资料多组间整体比较采用Kruskal-Wallis H检验。因无法观察同一个肝细胞核在不同时间点或酶处理前后的变化,因此不采用配对比较。所有统计采用双侧检验,P<0.05认为差异具有统计学意义。
研究结果：
第一部分：建立鸭乙型肝炎病毒后天感染模型
筛选出4只58日龄和2只1日龄血清DHBV DNA阳性鸭,感染率分别为20%和10%。
两组血清病毒阴性动物在2月龄或2日龄时静脉接种含1×108DHBV DNA病毒颗粒的血清。2月龄动物在接种后两个月内均未产生可检测的病毒血症,后天感染率为0,提示在成鸭中较难建立鸭乙型肝炎病毒后天感染模型。2日龄动物在接种后第14天,78.6%的动物可检出病毒血症,血清DHBV DNA水平在108copies/ml左右,随后观察的3个时间点,血清病毒载量均维持在107-109copies/ml之间。
第二部分：慢性感染状态下单个肝细胞核内DHBV DNA水平
成功建立单个核内DHBV DNA定量检测方法
将含有DHBV、HBV和HCV全基因组质粒进行荧光定量PCR扩增,发现除了DHBV质粒能成功扩增外,其余质粒均未产生荧光信号,无假阳性扩增,提示引物和探针的特异性好。
分别用1、2、10和20拷贝的PBS-DHBV1.2质粒进行荧光定量PCR扩增,发现除了1拷贝的质粒不能被有效扩增外,其余均能检出。两次独立重复试验均取得相一致结果：单个核内DHBV DNA最低定量检测下限(Low Limit of Detection, LLOD)为2拷贝。
分四批定量检测两只动物单个核内DHBV DNA水平,批间变异系数(CV%)分别为2.42%和6.92%。
单个核内DHBV DNA水平
慢性感染状态下,不是所有肝细胞核均受感染(63.3%-93.3%)。核与核之间病毒拷贝数差异较大(2-204)。Pearson相关性分析发现,核内病毒DNA平均拷贝数(7.57-57.67)与核内总的病毒载量(r=0.927,P<0.001)和血清病毒水平(r=0.605,P=0.049)呈正相关。
核内DHBV DNA水平与细胞周期有关
流式细胞仪对3只45日龄动物行细胞周期检测,发现分别有75%、81%、79%的肝细胞核处在G0/1期,15%、11%、10%的核处在S期,10%、8%、11%的核处于G2/M期。
细胞周期间病毒阳性核拷贝数整体比较有显著性差异(P<0.001),G0/1期最高,其次是G2/M和S期；阳性细胞核比率整体比较也有显著性差异(P<0.05),S期阳性细胞核比率最低,约一半的阳性核病毒拷贝数低于10。
PSAD酶处理对核内DHBV DNA水平影响较小
PSAD酶处理组与未处理组相比较,各动物病毒阳性核拷贝数均未见有显著变化：动物编号22(Z=-0.810, P=0.418),51(Z=-0.352, P=0.725),52(Z=-1.837, P=0.066),62(Z=-0.321, P=0.748),65(Z=1.041, P=0.298);各动物病毒阳性核比率也未发生统计学意义改变：动物编号22(χ2=0.098, P=0.075),51(χ2=0.351, P=0.554),52(χ2=0.741, P=0.389),62(χ2=0.480,P=0.488),65(χ2=1.071,P=0.301)。
核内DHBV DNA主要是以cccDNA的形式存在
Southern blot结果显示肝细胞核内DHBV DNA主要以cccDNA的形式存在,也可见有少量rcDNA;胞质中病毒DNA以rcDNA, dsDNA和ssDNA的形式存在。
第三部分：恩替卡韦治疗对单个核内DHBV DNA的影响
恩替卡韦治疗对病毒阳性肝细胞核比率的影响
恩替卡韦抗病毒治疗能显著降低病毒阳性肝细胞核比率(P<0.001)。阳性率下降主要发生在从基线至血清病毒DNA阴转时,延长治疗过程中阳性率下降仍较明显。
从基线至血清病毒DNA阴转时,恩替卡韦治疗组动物阳性肝细胞核比率明显降低(86.1%vs.50.6%,x2=52.580,P<0.001)；未治疗组阳性率未发生有统计学意义改变(70.0%vs.83.3%；x2=2.981,P=0.080)。
从血清病毒DNA阴转至治疗结束,恩替卡韦治疗组动物阳性肝细胞核比率明显降低(48.3%vs.25.8%；x2=13.019,P=0.001)；未治疗组阳性率未发生有统计学意义改变(83.3%vs.86.7%；x2=1.310,P=0.718)。
恩替卡韦治疗对单个核内DHBV DNA水平的影响
血清病毒DNA阴转时,恩替卡韦治疗组动物病毒阳性核拷贝数明显低于基线时水平(Z=-7.984,P=0.000)；未治疗组病毒阳性核拷贝数未发生统计学意义改变(t=0.313,P=0.755)。对每只动物进行分析,发现治疗组中除了9号(Z=-1.745,P=0.081),52号(t=1.479,P=0.148)动物病毒阳性核拷贝数无明显变化外,其余均明显降低。
从血清病毒DNA阴转至治疗结束,核内病毒DNA平均拷贝数缓慢降低,但个别核内病毒水平仍较高。
结论：
1.本研究成功建立单个核内DHBV DNA定量检测方法,该方法具有较理想的敏感性及特异性。
2.慢性感染状态下单个核内DHBV DNA拷贝数相差较大,核内DHBV DNA平均拷贝数与血清病毒水平及核内总的病毒载量正相关。
3.单个核内DHBV DNA水平与细胞周期状态有关。病毒在G0/1期复制活跃,其次是G2/M和S期。
4.抗病毒治疗能有效降低单个核内DHBV DNA水平及受感染肝细胞核数量。
Background and Aims:
Chronic infection is believed to be maintained by a replicative form of HBV DNA termed covalently closed circular DNA (cccDNA). During infection, HBV cccDNA accumulates in cell nuclei in which it persists as a stable episome and acts as a template for the transcription of viral genes, althougt its levels are much lower than rcDNA, it is essential to HBV replication and persistence of viral infection. With the knowledge gained from the molecular biology of HBV infection, it was shown that cccDNA plays a key role in viral persistence, viral reactivation after treatment withdrawal, and drug resistance.
cccDNA monitoring and the development of an accurate quantitative assay for cccDNA are becoming important in the understanding of HBV replication, viral clearance, antiviral efficiency evaluation and duration of antiviral treatment. Several studies have attempted to measure HBV cccDNA quantitatively, but nonspecific amplification of relaxed circular DNA is a major obstacle, which causes controversial cccDNA detection in peripheral blood monocyte cells (PBMC) or even in serum. Technical constraints and ethical considerations have also hampered the direct study of cccDNA maintenance and clearance mechanisms in patients to some extent.
For chronic duck hepatitis B virus (DHBV) infection, intranuclear viral DNA levels are10times lower than those in the cytoplasm, and predominantly consist of cccDNA. Thus, quantitative measurements and analysis of intranuclear viral genomes in single nuclei would show a well-defined image of the viral life cycle and can directly indicate cccDNA levels.
The aim of the present study was to develop a quantitative assay, then measure the content of DHBV DNA in single nuclei from DHBV-infected animals and observe intranuclear viral DNA kinetics under entecavir (ETV) therapy.
We first established the quantitative assay for analysis of viral DNA at single nuclei level and observe the kinetics of intranuclear viral DNA clearance undergoing entecavir therapy. By completing this study, it will help us well understanding the life cycle of hepadnavirus and the interaction between virus and host cells deeperly, also show a well-defined image of viral clearance at single nuclei level.Our study provides details on the viral life cycle and establishes a strong foundation for further research, including studies on intranuclear HBV DNA throughout the natural history of patients with chronic hepatitis B and evaluations of the efficacy of antiviral treatment in these patients.
Materials and Methods
Part1:Establishing the acquired duck hepatitis virus infection model
Twenty1-day-old and twenty58-day-old ducks were screened for the absence of DHBV infection by PCR amplification. All serum DHBV negative ducks were inoculated intravenously with108DHBV DNA-containing particles. Blood samples were taken at different time point. DHBV DNA was extracted from serum samples and amplified by quantitative and qualitative PCR.
Part2:Quantitation of single nuclear DHBV DNA levels in chronc DHBV-infected livers
1. Liver biopsy was performed in eleven45-day-old DHBV-infected ducks.
2. Establishing the method for quantitation of single nuclear DHBV DNA copy numbers.
Single nuclear DHBV DNA was quantitated by real-time PCR amplification. Sensitivity, specificity and interassay variability of intranuclear DHBV DNA quantitative assays were then evaluated.
Sensitivity of our quantitative real-time PCR assay was calibrated by DHBV plasmids (1,2,10, and20genomes). This assay achieved the lower limit of detection (LLOD) of2copies per nucleus, verified with duplicated assays.
Specificity was validated by the amplification control of plasmids containing HB V or HCV genomes.
With two ducks on four separate occasions, the interassay variability of this assay was evaluated by measuring average intranuclear DHBV DNA copy numbers.
3. Approximately5mg of frozen liver tissue was homogenized. Nuclei were collected by low-speed centrifugation, resuspended in lml of homogenization buffer, and subjected to one additional round of centrifugation. A suspension of nuclei was sorted into the individual wells of PCR plates. DNA was released from the individual nuclei by incubation with a proteinase K solution, then EcoR I was added. Total DNA released by proteinase K digestion and cut with EcoR I was then quantitated by real-time PCR amplification.
4. Total intranuclear DHBV DNA was extracted from105hepatocyte nuclei, which were sorted from nuclei suspension into a1.5ml Eppendorf centrifuge tube containing1ml PBS by using a high-speed cell sorter, and quantified.
5. Propidium iodide staining and sorting single nuclei in different cell cycle phases
Five hundred microliters of homogenization solution containing approximately105nuclei were incubated with15μl of propidium iodide staining solution (100ug/ml, Sigma-Aldrich, Rehovot, Israel) for at least30minutes at room temperature in the dark. The distribution of nuclei in the GO/1, S, or G2/M phases was determined by measuring nuclei DNA content with flow cytometry. Nuclei in different cell cycle phases were sorted into a96-well plate.
6. To exclude integrated viral DNA, DNA released from the individual nuclei was incubated with lunit of Plasmid-SafeTMTP-Dependent DNase (PSAD) at37℃for30min, after which the PSAD was inactivated by a further incubation at70℃for30min. Five units of EcoRI were then added and the samples were incubated at37℃for30min prior to PRC amplification. Viral DNA copies and the number of virus-positive nuclei were determined by real-time PCR. And the differences were compared between nuclei without or with PSAD treatment
7. Total liver DNA was extracted from150mg baseline liver tissue, and cell fractions (nuclei or cytoplasm) from300mg baseline liver tissue were prepared for DNA extraction using phenol-chloroform method. Total liver and cell-fraction DHBV DNA was detected using Southern blot procedures.
Part3:Effect of ETV therapy on intranuclear DHBV DNA levels
Active and chronic viral replication was confirmed with consecutive high DHBV DNA levels at41and43days of age in11ducks.On day0(45days of age), all ducks were weighed and subjected to a liver biopsy (first biopsy) as well as blood sampling. All ducks from the treated group underwent daily treatment with0.5mg of ETV by gavage until the end of the study. When serum DHBV DNA dropped below the lower limit of detection (LLOD) of the quantitative assay(103copies/ml) for2consecutive weeks, a second biopsy was conducted; parallel biopsies were performed in the untreated group. After16weeks further treatment, all animals were killed humanely and then autopsied; their livers were removed and cryo-stored for subsequent analysis. Between these two biopsies, animals were weighed and serum samples were taken once a week. After the second biopsy, blood samples were taken once every4weeks.
Kinetics of single nuclear DHBV DNA copies and the numbers of virus-positive nuclei will be observed.
Statistical analysis
The chi-square test, Student's t test, Pearson's correlation, Mann-Whitney U test and Kruskal-Wallis H test were used when appropriate. All statistical analyses were performed using SPSS version16.0(SPSS Inc., Chicago, IL, USA) and P<0.05was considered significant.
Results:
Part1:Establishing the acquired duck hepatitis virus infection model
Four58-day-old ducks and two1-day-old ducks were positive for serum DHBV DNA. The rates of the DHBV infection were20%and10%, respectively.
All animals with negative serum DHBV-DNA were inoculated intravenously with108DHBV DNA-containing particles on the following day or at the age of60days, respectively. In the adult ducks, there were no detectable viremia within2months post-inoculation, and the acquired infection rate was zero, it was difficult to establish the acquired infection model in the adult ducks; while78.6%of2-day-old ducklings had detectable viremia on day14post-inoculation. In the following time-point observed, serum DHBV DNA levels remained within the range of107-109genomes per ml.
Part2:Quantitation of single nuclear DHBV DNA levels in chronc DHBV-infected livers
Specificity, sensitivity, and interassay variability of intranuclear DHBV DNA quantitative assays.
After successful PCR performance, as showed by the amplification curve and the standard curve, specificity was validated by the negative amplification control of plasmids containing HBV or HCV genomes and none of the samples showed false-positive reactions
Sensitivity of our quantitative real-time PCR assay was calibrated by DHBV plasmids (1,2,10, and20genomes). This assay achieved the lower limit of detection (LLOD) of2copies per nucleus, verified with duplicated assays.
With two ducks on four separate occasions, the interassay variability of this assay was evaluated by measuring average intranuclear DHBV DNA copy numbers. The coefficients of variation were2.42%and6.92%, respectively.
Single nuclear DHBV DNA levels in chronic DHBV-infected ducks
In11ducks with active viral replication, the absolute intranuclear DHBV DNA copy numbers varied dramatically among the isolated nuclei (2-204copies/nuclei), and average copy numbers from individual animals distributed widely as well (7.57-57.67). As the lower limit of our assay was two copies per nucleus, it was possible that some nuclei contained exactly one DHBV DNA molecule or even no DHBV infection. The mean DHBV DNA copies were highly correlated with serum DHBV DNA titers (r=0.605, P=0.049) and total intranuclear DHBV DNA levels measured with105nuclei (r=0.927, P<0.001).
Intranuclear DHBV DNA levels correlated with cycle phase
The impact of the cell cycle phase on intranuclear DHBV DNA copy numbers was then further evaluated in three baseline ducks. Flow cytometry analysis for the individual animals showed that approximately75%,81%and79%of nuclei were arrested in the GO/1phase;15%,11%and10%in the S phase; and10%,8%and11%in the G2/M phase, respectively.
The differences of median DHBV DNA copies in virus-positive nuclei in the different cell cycle phase were significant (P=0.000), the median intranuclear viral DNA copies in the GO/1phase were significant higher than those in the G2/M and S phases; the differences of the ratio of virus-positive nuclei in the different cell cycle phase were also significant (P<0.05). In S phase, half of the virus-positive nuclei contained fewer than10copies.
PSAD treatments had little effects on intranuclear DHBV DNA quantitation
There was no significant change in DHBV DNA copies of virus-positive nuclei between nuclei without or with PSAD treatment in duck22(Z=-0.810, P=0.418),51(Z=-0.352, P=0.725),52(Z=-1.837, P=0.066),62(Z=-0.321, P=0.748) and65(Z-1.041, P=0.298); there was also no significant change in the percentages of virus-positive nuclei between nuclei without or with PSAD treatment in duck22(χ2=0.098, P=0.075),51(χ2=0.351, P=0.554),52(χ2=0.741, P=0.389),62(χ=0.480, P=0.488) and65(χ2=1.071, P=0.301).
cccDNA is the predominant form of intranuclear DHBV DNA
Viral DNA in the cytoplasmic fraction existed as rcDNA, double-stranded DNA and single-stranded DNA forms; whereas much of the viral DNA in the nuclear fraction migrated to the cccDNA position, and a less intense band was presented at the rcDNA position. These results indicated that intranuclear DHBV DNA mainly comprised of cccDNA form and contained less rcDNA.
Part3:Effect of ETV therapy on intranuclear DHBV DNA levels
Effect of ETV therapy on the ratio of virus-positive nuclei
Six45-day-old ducks with active viral replication were administered with ETV. Their serum DHBV DNA levels decreased dramatically compared with the untreated ducks during ETV antiviral therapy.
From baseline to the end of the study, entecavir would significantly reduced the numbers of virus-positive nuclei (P<0.01). This trend was very obvious from baseline to the time when serum DHBV DNA became negative, while the reduction was less obvious from then to the end of the observation.
From baseline to the time when serum DHBV DNA became negative, the percentages of virus-positive nuclei in the ETV-treated ducks decreased (86.1%vs.50.6%, P<0.001), no statistically significant change was found in the untreated animals (70%vs.83.3%, P=0.084).
From the time when serum DHBV DNA became negative to the end of the observation, the percentages of virus-positive nuclei decreased in the treated ducks (48.3%vs.25.8%, P=0.001); there was no significant change in the ratio (83.3%vs.86.7%, P=0.718) of virus-positive nuclei in the untreated group.
Effect of ETV therapy on the levels of single nuclear DHBV DNA
From baseline to the time when serum DHBV DNA became negative, DHBV DNA copies of virus-positive nuclei decreased significantly in the ETV-treated group (Z=-7.984, P=0.000); no statistically significant change was observed in the untreated group (t=0.313, P=0.755). With the exception of duck9(Z=-1.745, P=0.081) and52(t=1.479,0.148), DHBV DNA copies of virus-positive nuclei all declined significantly in other animals.
From the time when serum DHBV DNA became negative to the end of the observation, although the average viral DNA copies declined slowly, viral copy numbers in few virus-positive nuclei at the end of the study were greater than those from the second biopsy, which was taken at the point of complete serum viral suppression.
Conclusion
1. We have successfully established a quantitative assay to measure intranuclear DHBV DNA in single nuclei with high specificity, sensitivity.
2. The intranuclear viral DNA copy numbers varied dramatically in11ducks with active viral replication. Average intranuclear DHBV DNA copies positively correlated with total intranuclear and serum viral DNA levels.
3. The intranuclear DHBV DNA copies were regulated by the cell cycle status, and were greater in the GO/1than those in the G2/M and S phases.
4. Entecavir therapy could effectively reduce the intranuclear DHBV levels and the numbers of virus-infected nuclei.
共价闭合环状DNA (covalently closed circular DNA, cccDNA)是乙型肝炎病毒(Hepatitis B virus, HBV)的复制模板,长期稳定的以微染色体的形式存在于受感染的肝细胞核内,虽其含量远低于HBV存在的一般形式松弛环状DNA(relaxed circular DNA, rcDNA),但对HBV的复制和感染状态的建立具有举足轻重的意义。cccDNA是HBV感染状态的持续、停用抗病毒药物后病情反复及药物耐药的关键因素,因此只有清除或有效降低细胞核内的cccDNA,才能彻底消除乙肝患者病毒携带状态,或使病情趋于稳定。
cccDNA的定量检测对于理解HBV的复制和清除,评估抗病毒药物的疗效和疗程等具有重要的意义；但rcDNA或双链线性DNA (double strand DNA, dsDNA)的非特异性扩增仍然是目前要面临的主要问题。检测方法不成熟和肝组织来源困难在一定程度上阻碍了对乙肝患者cccDNA的直接研究。
在鸭乙型肝炎病毒(Duck hepatitis B virus, DHBV)感染的鸭肝细胞,胞核内病毒DNA主要是以cccDNA为主,而胞浆内作为cccDNA前体的rcDNA可能有其量的10倍。因此定量检测核内病毒DNA可较直接反映cccDNA的水平。
本研究将在单个肝细胞核的层面上定量检测慢性DHBV感染状态下核内病毒DNA水平及其影响因素；通过恩替卡韦短期抑制病毒复制,观察血清DHBVDNA阴转时及阴转后单个肝细胞核内病毒水平及受感染肝细胞核数的动态变化。
我们的研究首次提出了在单个肝细胞核的水平上分析核内病毒DNA,并通过恩替卡韦抑制病毒复制观察核内病毒DNA的清除动力学。完成这一研究会帮助我们更加深入理解嗜肝DNA病毒的生活周期,病毒与宿主细胞间的相互作用,药物作用下单个核内病毒DNA的清除规律,同时也将为下一步研究慢乙肝患者核内HBV DNA水平及抗病毒疗效评估等奠定基础。
研究方法：
第一部分：建立鸭乙型肝炎病毒后天感染模型
筛选1和58日龄广东樱桃谷鸭各20只,在2日及2月龄时病毒阴性动物静脉接种含1×108DHBV DNA病毒颗粒的血清。接种后不同时间点采血,抽提DNA,PCR定性及定量检测外周血病毒DNA水平。
第二部分：慢性感染状态下单个肝细胞核内DHBV DNA水平
1.对纳入的11只45日龄慢性DHBV感染鸭行肝活检手术
2.建立单个核内DHBV DNA定量检测方法
采用Taqman探针荧光定量PCR法检测单个核内DHBV DNA水平,并验证该方法的敏感性、特异性及批间差异：
已知浓度的标准质粒(PBS-DHBV1.2)倍比稀释为1、10、100、1000和2、20、200、2000copies/μl。按照50μl的反应体系分别向反应孔内加入1、10、100、1000和2、20、200、2000拷贝数的PBS-DHBV1.2质粒进行real-time PCR定量扩增,得到扩增曲线和标准曲线,确定方法的敏感性(最低检测下限)；
分别用含有DHBV、HBV及HCV全基因组的质粒进行扩增,验证引物和探针的特异性；
分四批定量检测两只动物单个核内DHBV DNA水平,计算核内病毒拷贝数的批间差异,用变异系数(CV%)表示。
3.匀浆研磨5mg肝组织、低速离心、匀浆液充分洗涤得到肝细胞核悬液、通过流式细胞仪分选得到单个细胞核；单个细胞核经蛋白酶K消化、EcoRI酶切后,采用荧光定量PCR法检测核内病毒DNA水平。
4.流式细胞仪分选105个肝细胞核至1ml的PBS溶液中、提取总的核内DNA,采用荧光定量PCR法检测总的核内病毒DNA水平。
5.细胞周期检测并分选处在不同周期的单个核
500μ1的肝细胞核悬液(约1×105个核)中加入15μl PI(100μg/ml)染色液,避光室温孵育至少30min;流式细胞仪将根据核内DNA的量确定细胞周期的分布状况,并分选处在不同细胞周期的单个核至96孔板中。
6.明确整合的病毒DNA影响
质粒安全ATP依赖的DNA酶(PSAD)可以选择性消化线性DNA(即整合的片段),对cccDNA和rcDNA无影响。单个核内病毒DNA在1个单位PSAD酶的作用下37℃孵育30min,然后70℃、30min灭活该酶;再加入5个单位的EcoR I,37℃孵育30min。采用荧光定量PCR法检测单个核内病毒DNA水平,确定病毒阳性细胞核数,比较PSAD处理组与未处理组间的差异。
7.病毒DNA在肝内的存在形式
采用酚-氯仿抽提法提取肝细胞核、细胞质及肝细胞内的总DNA, Southern blot印迹杂交检测病毒DNA的存在形式。
第三部分：恩替卡韦治疗对单个核内DHBV DNA的影响
实验分组：将11只45日龄慢性DHBV感染鸭随机分两组：恩替卡韦治疗组(n=6)和未治疗组(n=5)。
治疗组每只动物每日接受0.5mg恩替卡韦抗病毒,血清DHBV DNA阴转时,行第二次肝活检；阴转后继续给药治疗16周,治疗结束时处死所用动物,取出肝组织标本。
观察单个核内DHBV DNA水平及受感染肝细胞核数的动态变化。
统计学分析：
所有数据采用SPSS16.0进行处理。计数资料组间比较采用Pearson's chi-square检验,对理论频数小于5的四格表资料采用Fisher精确概率法。连续变量之间的相关性应用Pearson方法。计量资料若满足正态分布或方差齐性采用参数检验方法(两独立样本t检验)；计量资料若不满足正态分布及方差不齐则采用非参数检验方法(Mann-Whitney U检验)。计量资料多组间整体比较采用Kruskal-Wallis H检验。因无法观察同一个肝细胞核在不同时间点或酶处理前后的变化,因此不采用配对比较。所有统计采用双侧检验,P<0.05认为差异具有统计学意义。
研究结果：
第一部分：建立鸭乙型肝炎病毒后天感染模型
筛选出4只58日龄和2只1日龄血清DHBV DNA阳性鸭,感染率分别为20%和10%。
两组血清病毒阴性动物在2月龄或2日龄时静脉接种含1×108DHBV DNA病毒颗粒的血清。2月龄动物在接种后两个月内均未产生可检测的病毒血症,后天感染率为0,提示在成鸭中较难建立鸭乙型肝炎病毒后天感染模型。2日龄动物在接种后第14天,78.6%的动物可检出病毒血症,血清DHBV DNA水平在108copies/ml左右,随后观察的3个时间点,血清病毒载量均维持在107-109copies/ml之间。
第二部分：慢性感染状态下单个肝细胞核内DHBV DNA水平
成功建立单个核内DHBV DNA定量检测方法
将含有DHBV、HBV和HCV全基因组质粒进行荧光定量PCR扩增,发现除了DHBV质粒能成功扩增外,其余质粒均未产生荧光信号,无假阳性扩增,提示引物和探针的特异性好。
分别用1、2、10和20拷贝的PBS-DHBV1.2质粒进行荧光定量PCR扩增,发现除了1拷贝的质粒不能被有效扩增外,其余均能检出。两次独立重复试验均取得相一致结果：单个核内DHBV DNA最低定量检测下限(Low Limit of Detection, LLOD)为2拷贝。
分四批定量检测两只动物单个核内DHBV DNA水平,批间变异系数(CV%)分别为2.42%和6.92%。
单个核内DHBV DNA水平
慢性感染状态下,不是所有肝细胞核均受感染(63.3%-93.3%)。核与核之间病毒拷贝数差异较大(2-204)。Pearson相关性分析发现,核内病毒DNA平均拷贝数(7.57-57.67)与核内总的病毒载量(r=0.927,P<0.001)和血清病毒水平(r=0.605,P=0.049)呈正相关。
核内DHBV DNA水平与细胞周期有关
流式细胞仪对3只45日龄动物行细胞周期检测,发现分别有75%、81%、79%的肝细胞核处在G0/1期,15%、11%、10%的核处在S期,10%、8%、11%的核处于G2/M期。
细胞周期间病毒阳性核拷贝数整体比较有显著性差异(P<0.001),G0/1期最高,其次是G2/M和S期；阳性细胞核比率整体比较也有显著性差异(P<0.05),S期阳性细胞核比率最低,约一半的阳性核病毒拷贝数低于10。
PSAD酶处理对核内DHBV DNA水平影响较小
PSAD酶处理组与未处理组相比较,各动物病毒阳性核拷贝数均未见有显著变化：动物编号22(Z=-0.810, P=0.418),51(Z=-0.352, P=0.725),52(Z=-1.837, P=0.066),62(Z=-0.321, P=0.748),65(Z=1.041, P=0.298);各动物病毒阳性核比率也未发生统计学意义改变：动物编号22(χ2=0.098, P=0.075),51(χ2=0.351, P=0.554),52(χ2=0.741, P=0.389),62(χ2=0.480,P=0.488),65(χ2=1.071,P=0.301)。
核内DHBV DNA主要是以cccDNA的形式存在
Southern blot结果显示肝细胞核内DHBV DNA主要以cccDNA的形式存在,也可见有少量rcDNA;胞质中病毒DNA以rcDNA, dsDNA和ssDNA的形式存在。
第三部分：恩替卡韦治疗对单个核内DHBV DNA的影响
恩替卡韦治疗对病毒阳性肝细胞核比率的影响
恩替卡韦抗病毒治疗能显著降低病毒阳性肝细胞核比率(P<0.001)。阳性率下降主要发生在从基线至血清病毒DNA阴转时,延长治疗过程中阳性率下降仍较明显。
从基线至血清病毒DNA阴转时,恩替卡韦治疗组动物阳性肝细胞核比率明显降低(86.1%vs.50.6%,x2=52.580,P<0.001)；未治疗组阳性率未发生有统计学意义改变(70.0%vs.83.3%；x2=2.981,P=0.080)。
从血清病毒DNA阴转至治疗结束,恩替卡韦治疗组动物阳性肝细胞核比率明显降低(48.3%vs.25.8%；x2=13.019,P=0.001)；未治疗组阳性率未发生有统计学意义改变(83.3%vs.86.7%；x2=1.310,P=0.718)。
恩替卡韦治疗对单个核内DHBV DNA水平的影响
血清病毒DNA阴转时,恩替卡韦治疗组动物病毒阳性核拷贝数明显低于基线时水平(Z=-7.984,P=0.000)；未治疗组病毒阳性核拷贝数未发生统计学意义改变(t=0.313,P=0.755)。对每只动物进行分析,发现治疗组中除了9号(Z=-1.745,P=0.081),52号(t=1.479,P=0.148)动物病毒阳性核拷贝数无明显变化外,其余均明显降低。
从血清病毒DNA阴转至治疗结束,核内病毒DNA平均拷贝数缓慢降低,但个别核内病毒水平仍较高。
结论：
1.本研究成功建立单个核内DHBV DNA定量检测方法,该方法具有较理想的敏感性及特异性。
2.慢性感染状态下单个核内DHBV DNA拷贝数相差较大,核内DHBV DNA平均拷贝数与血清病毒水平及核内总的病毒载量正相关。
3.单个核内DHBV DNA水平与细胞周期状态有关。病毒在G0/1期复制活跃,其次是G2/M和S期。
4.抗病毒治疗能有效降低单个核内DHBV DNA水平及受感染肝细胞核数量。
Background and Aims:
Chronic infection is believed to be maintained by a replicative form of HBV DNA termed covalently closed circular DNA (cccDNA). During infection, HBV cccDNA accumulates in cell nuclei in which it persists as a stable episome and acts as a template for the transcription of viral genes, althougt its levels are much lower than rcDNA, it is essential to HBV replication and persistence of viral infection. With the knowledge gained from the molecular biology of HBV infection, it was shown that cccDNA plays a key role in viral persistence, viral reactivation after treatment withdrawal, and drug resistance.
cccDNA monitoring and the development of an accurate quantitative assay for cccDNA are becoming important in the understanding of HBV replication, viral clearance, antiviral efficiency evaluation and duration of antiviral treatment. Several studies have attempted to measure HBV cccDNA quantitatively, but nonspecific amplification of relaxed circular DNA is a major obstacle, which causes controversial cccDNA detection in peripheral blood monocyte cells (PBMC) or even in serum. Technical constraints and ethical considerations have also hampered the direct study of cccDNA maintenance and clearance mechanisms in patients to some extent.
For chronic duck hepatitis B virus (DHBV) infection, intranuclear viral DNA levels are10times lower than those in the cytoplasm, and predominantly consist of cccDNA. Thus, quantitative measurements and analysis of intranuclear viral genomes in single nuclei would show a well-defined image of the viral life cycle and can directly indicate cccDNA levels.
The aim of the present study was to develop a quantitative assay, then measure the content of DHBV DNA in single nuclei from DHBV-infected animals and observe intranuclear viral DNA kinetics under entecavir (ETV) therapy.
We first established the quantitative assay for analysis of viral DNA at single nuclei level and observe the kinetics of intranuclear viral DNA clearance undergoing entecavir therapy. By completing this study, it will help us well understanding the life cycle of hepadnavirus and the interaction between virus and host cells deeperly, also show a well-defined image of viral clearance at single nuclei level.Our study provides details on the viral life cycle and establishes a strong foundation for further research, including studies on intranuclear HBV DNA throughout the natural history of patients with chronic hepatitis B and evaluations of the efficacy of antiviral treatment in these patients.
Materials and Methods
Part1:Establishing the acquired duck hepatitis virus infection model
Twenty1-day-old and twenty58-day-old ducks were screened for the absence of DHBV infection by PCR amplification. All serum DHBV negative ducks were inoculated intravenously with108DHBV DNA-containing particles. Blood samples were taken at different time point. DHBV DNA was extracted from serum samples and amplified by quantitative and qualitative PCR.
Part2:Quantitation of single nuclear DHBV DNA levels in chronc DHBV-infected livers
1. Liver biopsy was performed in eleven45-day-old DHBV-infected ducks.
2. Establishing the method for quantitation of single nuclear DHBV DNA copy numbers.
Single nuclear DHBV DNA was quantitated by real-time PCR amplification. Sensitivity, specificity and interassay variability of intranuclear DHBV DNA quantitative assays were then evaluated.
Sensitivity of our quantitative real-time PCR assay was calibrated by DHBV plasmids (1,2,10, and20genomes). This assay achieved the lower limit of detection (LLOD) of2copies per nucleus, verified with duplicated assays.
Specificity was validated by the amplification control of plasmids containing HB V or HCV genomes.
With two ducks on four separate occasions, the interassay variability of this assay was evaluated by measuring average intranuclear DHBV DNA copy numbers.
3. Approximately5mg of frozen liver tissue was homogenized. Nuclei were collected by low-speed centrifugation, resuspended in lml of homogenization buffer, and subjected to one additional round of centrifugation. A suspension of nuclei was sorted into the individual wells of PCR plates. DNA was released from the individual nuclei by incubation with a proteinase K solution, then EcoR I was added. Total DNA released by proteinase K digestion and cut with EcoR I was then quantitated by real-time PCR amplification.
4. Total intranuclear DHBV DNA was extracted from105hepatocyte nuclei, which were sorted from nuclei suspension into a1.5ml Eppendorf centrifuge tube containing1ml PBS by using a high-speed cell sorter, and quantified.
5. Propidium iodide staining and sorting single nuclei in different cell cycle phases
Five hundred microliters of homogenization solution containing approximately105nuclei were incubated with15μl of propidium iodide staining solution (100ug/ml, Sigma-Aldrich, Rehovot, Israel) for at least30minutes at room temperature in the dark. The distribution of nuclei in the GO/1, S, or G2/M phases was determined by measuring nuclei DNA content with flow cytometry. Nuclei in different cell cycle phases were sorted into a96-well plate.
6. To exclude integrated viral DNA, DNA released from the individual nuclei was incubated with lunit of Plasmid-SafeTMTP-Dependent DNase (PSAD) at37℃for30min, after which the PSAD was inactivated by a further incubation at70℃for30min. Five units of EcoRI were then added and the samples were incubated at37℃for30min prior to PRC amplification. Viral DNA copies and the number of virus-positive nuclei were determined by real-time PCR. And the differences were compared between nuclei without or with PSAD treatment
7. Total liver DNA was extracted from150mg baseline liver tissue, and cell fractions (nuclei or cytoplasm) from300mg baseline liver tissue were prepared for DNA extraction using phenol-chloroform method. Total liver and cell-fraction DHBV DNA was detected using Southern blot procedures.
Part3:Effect of ETV therapy on intranuclear DHBV DNA levels
Active and chronic viral replication was confirmed with consecutive high DHBV DNA levels at41and43days of age in11ducks.On day0(45days of age), all ducks were weighed and subjected to a liver biopsy (first biopsy) as well as blood sampling. All ducks from the treated group underwent daily treatment with0.5mg of ETV by gavage until the end of the study. When serum DHBV DNA dropped below the lower limit of detection (LLOD) of the quantitative assay(103copies/ml) for2consecutive weeks, a second biopsy was conducted; parallel biopsies were performed in the untreated group. After16weeks further treatment, all animals were killed humanely and then autopsied; their livers were removed and cryo-stored for subsequent analysis. Between these two biopsies, animals were weighed and serum samples were taken once a week. After the second biopsy, blood samples were taken once every4weeks.
Kinetics of single nuclear DHBV DNA copies and the numbers of virus-positive nuclei will be observed.
Statistical analysis
The chi-square test, Student's t test, Pearson's correlation, Mann-Whitney U test and Kruskal-Wallis H test were used when appropriate. All statistical analyses were performed using SPSS version16.0(SPSS Inc., Chicago, IL, USA) and P<0.05was considered significant.
Results:
Part1:Establishing the acquired duck hepatitis virus infection model
Four58-day-old ducks and two1-day-old ducks were positive for serum DHBV DNA. The rates of the DHBV infection were20%and10%, respectively.
All animals with negative serum DHBV-DNA were inoculated intravenously with108DHBV DNA-containing particles on the following day or at the age of60days, respectively. In the adult ducks, there were no detectable viremia within2months post-inoculation, and the acquired infection rate was zero, it was difficult to establish the acquired infection model in the adult ducks; while78.6%of2-day-old ducklings had detectable viremia on day14post-inoculation. In the following time-point observed, serum DHBV DNA levels remained within the range of107-109genomes per ml.
Part2:Quantitation of single nuclear DHBV DNA levels in chronc DHBV-infected livers
Specificity, sensitivity, and interassay variability of intranuclear DHBV DNA quantitative assays.
After successful PCR performance, as showed by the amplification curve and the standard curve, specificity was validated by the negative amplification control of plasmids containing HBV or HCV genomes and none of the samples showed false-positive reactions
Sensitivity of our quantitative real-time PCR assay was calibrated by DHBV plasmids (1,2,10, and20genomes). This assay achieved the lower limit of detection (LLOD) of2copies per nucleus, verified with duplicated assays.
With two ducks on four separate occasions, the interassay variability of this assay was evaluated by measuring average intranuclear DHBV DNA copy numbers. The coefficients of variation were2.42%and6.92%, respectively.
Single nuclear DHBV DNA levels in chronic DHBV-infected ducks
In11ducks with active viral replication, the absolute intranuclear DHBV DNA copy numbers varied dramatically among the isolated nuclei (2-204copies/nuclei), and average copy numbers from individual animals distributed widely as well (7.57-57.67). As the lower limit of our assay was two copies per nucleus, it was possible that some nuclei contained exactly one DHBV DNA molecule or even no DHBV infection. The mean DHBV DNA copies were highly correlated with serum DHBV DNA titers (r=0.605, P=0.049) and total intranuclear DHBV DNA levels measured with105nuclei (r=0.927, P<0.001).
Intranuclear DHBV DNA levels correlated with cycle phase
The impact of the cell cycle phase on intranuclear DHBV DNA copy numbers was then further evaluated in three baseline ducks. Flow cytometry analysis for the individual animals showed that approximately75%,81%and79%of nuclei were arrested in the GO/1phase;15%,11%and10%in the S phase; and10%,8%and11%in the G2/M phase, respectively.
The differences of median DHBV DNA copies in virus-positive nuclei in the different cell cycle phase were significant (P=0.000), the median intranuclear viral DNA copies in the GO/1phase were significant higher than those in the G2/M and S phases; the differences of the ratio of virus-positive nuclei in the different cell cycle phase were also significant (P<0.05). In S phase, half of the virus-positive nuclei contained fewer than10copies.
PSAD treatments had little effects on intranuclear DHBV DNA quantitation
There was no significant change in DHBV DNA copies of virus-positive nuclei between nuclei without or with PSAD treatment in duck22(Z=-0.810, P=0.418),51(Z=-0.352, P=0.725),52(Z=-1.837, P=0.066),62(Z=-0.321, P=0.748) and65(Z-1.041, P=0.298); there was also no significant change in the percentages of virus-positive nuclei between nuclei without or with PSAD treatment in duck22(χ2=0.098, P=0.075),51(χ2=0.351, P=0.554),52(χ2=0.741, P=0.389),62(χ=0.480, P=0.488) and65(χ2=1.071, P=0.301).
cccDNA is the predominant form of intranuclear DHBV DNA
Viral DNA in the cytoplasmic fraction existed as rcDNA, double-stranded DNA and single-stranded DNA forms; whereas much of the viral DNA in the nuclear fraction migrated to the cccDNA position, and a less intense band was presented at the rcDNA position. These results indicated that intranuclear DHBV DNA mainly comprised of cccDNA form and contained less rcDNA.
Part3:Effect of ETV therapy on intranuclear DHBV DNA levels
Effect of ETV therapy on the ratio of virus-positive nuclei
Six45-day-old ducks with active viral replication were administered with ETV. Their serum DHBV DNA levels decreased dramatically compared with the untreated ducks during ETV antiviral therapy.
From baseline to the end of the study, entecavir would significantly reduced the numbers of virus-positive nuclei (P<0.01). This trend was very obvious from baseline to the time when serum DHBV DNA became negative, while the reduction was less obvious from then to the end of the observation.
From baseline to the time when serum DHBV DNA became negative, the percentages of virus-positive nuclei in the ETV-treated ducks decreased (86.1%vs.50.6%, P<0.001), no statistically significant change was found in the untreated animals (70%vs.83.3%, P=0.084).
From the time when serum DHBV DNA became negative to the end of the observation, the percentages of virus-positive nuclei decreased in the treated ducks (48.3%vs.25.8%, P=0.001); there was no significant change in the ratio (83.3%vs.86.7%, P=0.718) of virus-positive nuclei in the untreated group.
Effect of ETV therapy on the levels of single nuclear DHBV DNA
From baseline to the time when serum DHBV DNA became negative, DHBV DNA copies of virus-positive nuclei decreased significantly in the ETV-treated group (Z=-7.984, P=0.000); no statistically significant change was observed in the untreated group (t=0.313, P=0.755). With the exception of duck9(Z=-1.745, P=0.081) and52(t=1.479,0.148), DHBV DNA copies of virus-positive nuclei all declined significantly in other animals.
From the time when serum DHBV DNA became negative to the end of the observation, although the average viral DNA copies declined slowly, viral copy numbers in few virus-positive nuclei at the end of the study were greater than those from the second biopsy, which was taken at the point of complete serum viral suppression.
Conclusion
1. We have successfully established a quantitative assay to measure intranuclear DHBV DNA in single nuclei with high specificity, sensitivity.
2. The intranuclear viral DNA copy numbers varied dramatically in11ducks with active viral replication. Average intranuclear DHBV DNA copies positively correlated with total intranuclear and serum viral DNA levels.
3. The intranuclear DHBV DNA copies were regulated by the cell cycle status, and were greater in the GO/1than those in the G2/M and S phases.
4. Entecavir therapy could effectively reduce the intranuclear DHBV levels and the numbers of virus-infected nuclei.
引文
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[2]. Liang X, Bi S, Yang W, Wang L, Cui G, Cui F, Zhang Y, et al. Epidemiological serosurvey of hepatitis B in China--declining HBV prevalence due to hepatitis B vaccination. Vaccine 2009;27:6550-6557.
[3]. Liang X, Bi S, Yang W, Wang L, Cui G, Cui F, Zhang Y, et al. Evaluation of the impact of hepatitis B vaccination among children born during 1992-2005 in China. J Infect Dis 2009;200:39-47.
[4]. Lu FM, Zhuang H. Management of hepatitis B in China. Chin Med J (Engl) 2009;122:3-4.
[5]. Hoofnagle JH, Doo E, Liang TJ, Fleischer R, Lok AS. Management of hepatitis B:summary of a clinical research workshop. Hepatology 2007;45:1056-1075.
[6]. Belloni L, Allweiss L, Guerrieri F, Pediconi N, Volz T, Pollicino T, Petersen J, et al. IFN-alpha inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome. J Clin Invest 2012.
[7]. Zoulim F. New insight on hepatitis B virus persistence from the study of intrahepatic viral cccDNA. Journal of Hepatology 2005;42:302-308.
[8]. Tuttleman JS, Pourcel C, Summers J. Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell 1986;47:451-460.
[9]. Seeger C, Mason WS. Hepatitis B virus biology. Microbiology and molecular biology reviews 2000;64:51.
[10]. Levrero M, Pollicino T, Petersen J, Belloni L, Raimondo G, Dandri M. Control of cccDNA function in hepatitis B virus infection. Journal of hepatology 2009;51:581-592.
[11]. Mason WS, Halpern MS, England JM, Seal G, Egan J, Coates L, Aldrich C, et al. Experimental transmission of duck hepatitis B virus. Virology 1983;131:375-384.
[12]. Kock J, Schlicht HJ. Analysis of the earliest steps of hepadnavirus replication: genome repair after infectious entry into hepatocytes does not depend on viral polymerase activity. J Virol 1993;67:4867-4874.
[13]. Habig JW, Loeb DD. Template switches during plus-strand DNA synthesis of duck hepatitis B virus are influenced by the base composition of the minus-strand terminal redundancy. J Virol 2003;77:12412-12420.
[14]. Liu N, Ji L, Maguire ML, Loeb DD. cis-Acting sequences that contribute to the synthesis of relaxed-circular DNA of human hepatitis B virus. J Virol 2004;78:642-649.
[15]. Ganem D, Prince AM. Hepatitis B virus infection--natural history and clinical consequences. N Engl J Med 2004;350:1118-1129.
[16]. Tagawa M, Omata M, Okuda K. Appearance of viral RNA transcripts in the early stage of duck hepatitis B virus infection. Virology 1986; 152:477-482.
[17]. Yang W, Summers J. Illegitimate replication of linear hepadnavirus DNA through nonhomologous recombination. J Virol 1995;69:4029-4036.
[18]. Staprans S, Loeb DD, Ganem D. Mutations affecting hepadnavirus plus-strand DNA synthesis dissociate primer cleavage from translocation and reveal the origin of linear viral DNA. J Virol 1991;65:1255-1262.
[19]. Wu TT, Coates L, Aldrich CE, Summers J, Mason WS. In hepatocytes infected with duck hepatitis B virus, the template for viral RNA synthesis is amplified by an intracellular pathway. Virology 1990;175:255-261.
[20]. Summers J, Smith PM, Huang MJ, Yu MS. Morphogenetic and regulatory effects of mutations in the envelope proteins of an avian hepadnavirus. J Virol 1991;65:1310-1317.
[21]. Civitico GM, Locarnini SA. The half-life of duck hepatitis B virus supercoiled DNA in congenitally infected primary hepatocyte cultures. Virology 1994;203:81-89.
[22]. Delaney WT, Miller TG, Isom HC. Use of the hepatitis B virus recombinant baculovirus-HepG2 system to study the effects of (-)-beta-2',3'-dideoxy-31-thiacytidine on replication of hepatitis B virus and accumulation of covalently closed circular DNA. Antimicrob Agents Chemother 1999;43:2017-2026.
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