| 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 MethodsPart1:Establishing the acquired duck hepatitis virus infection modelTwenty1-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 livers1. 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 phasesFive 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 treatment7. 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 levelsActive 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 analysisThe 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 modelFour58-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 liversSpecificity, 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 reactionsSensitivity 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 ducksIn11ducks 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 phaseThe 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 quantitationThere 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 DNAViral 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 levelsEffect of ETV therapy on the ratio of virus-positive nucleiSix45-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 DNAFrom 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. Conclusion1. 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. |
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