Hepatocyte turnover in transient and chronic hepadnavirus infections

Summary.  Hepatocyte turnover appears to be an important feature in the resolution of transient and progression of chronic hepadnavirus infections. Hepatocyte death, initiated through attack by antiviral cytotoxic T-lymphocytes (CTL), and compensatory hepatocyte proliferation, are both believed to be major contributing factors in the loss of virus DNA during immune resolution of transient infections. Noncytopathic curing of hepatocytes is also suggested to occur, though this mechanism does not prevent the death of large numbers of hepatocytes. Hepatocyte death, proliferation and curing are also important features of chronic infections, though the outcomes are different. In particular, immune selection due to persistent attack by antiviral CTL is thought to play a role in the emergence of hepatocytes infected with mutant strains of hepatitis B virus (HBV) (e.g. HBV e antigen-negative strains) and in the emergence of hepatocytes that appear refractory to HBV infection. In both instances, clonal expansion of subpopulations of hepatocytes may be inferred to have taken place. Interestingly, foci of altered hepatocytes and hepatocellular carcinomas (HCC) typically do no support virus replication. Thus, immune selection of hepatocytes by antiviral CTL, by inducing clonal expansion, may also play an important role in the progression to HCC. In this review, we discuss the evidence in support of roles for hepatocyte turnover in the resolution of transient and progression of chronic HBV infections.     

Compartmental HBV evolution and replication in liver and extrahepatic sites after nucleos/tide analogue therapy in chronic hepatitis B carriers

BackgroundHepatitis B virus (HBV) variants are associated with nucleos/tide analogue (NA) response and liver disease but it is unknown whether NA influences extrahepatic HBV persistence.ObjectivesTo investigate HBV replication and genetic evolution in hepatic and extrahepatic sites of chronic hepatitis B (CHB) before and after NA therapy.Study DesignA total of 13 paired plasma, peripheral blood mononuclear cells (PBMC), were collected from chronic HBV carriers at baseline and after a median 53 weeks NA therapy as well as liver biopsy (N = 7 baseline, N = 5 follow-up). HBV covalently closed circular DNA (cccDNA) and messenger (m) RNA in liver and PBMC were analyzed. HBV polymerase (P)/surface (S), basal core promoter (BCP)/pre-core (PC)/C gene clonal sequencing was done in plasma, peripheral blood mononuclear cells (PBMC), and liver.ResultsCompare to baseline, at ∼53 weeks follow-up, there was no significant change in HBV cccDNA levels in liver (0.2–0.08 copies/hepatocyte, p > 0.05) or in PBMC 0.003–0.02 copies/PBMC, p > 0.05), and HBV mRNA remained detectable in both sites. At baseline, BCP variants were higher in PBMC vs. liver and plasma. After therapy, drug resistant (DR) and immune escape (IE) variants increased in liver but IE and PC variants were more frequent in PBMC. HBV P/S diversity was significantly higher in PBMC compared to plasma.ConclusionContinuous HBV replication occurs in liver and PBMC and shows compartmentalized evolution under selective pressure of potent NA therapy.     

Detection of Hepatitis B Virus Covalently Closed Circular DNA in the Plasma of Iranian HBeAg-Negative Patients With Chronic Hepatitis B

Background:

Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is a marker of HBV replication in the liver of patients infected with HBV.

Objectives:

This study aimed to investigate the association between the presence of cccDNA in the plasma samples of Iranian treatment-naive patients with chronic hepatitis B infection and HBV viral load and HBsAg levels.

Patients and Methods:

From April 2012 to May 2015, 106 treatment-naive patients with chronic hepatitis B infection were enrolled in this cross-sectional study. The HBsAg titer was measured by the Roche HBsAg II assay on the Cobas e411 system, and HBV DNA quantitation was performed using the COBAS TaqMan 48 kit. Real-time polymerase chain reaction was performed for the detection of HBV cccDNA.

Results:

The mean (SD) age of the patients was 41.1 ± 12.4 years (range, 20 - 62 years). From a total of 106 study participants, 67 (63.2%) were males. The HBV cccDNA was detected in plasma specimens in 19 (17.9%) out of the total 106 patients, and a significant relationship was found between the presence of cccDNA in plasma sample of males (23.9%) and females (7.7%) (P = 0.039). Also, a significant correlation was found between the presence of cccDNA in plasma sample of the patients and HBV viral load level (P < 0.0001) and HBsAg titer (P = 0.0043).

Conclusions:

This study showed that cccDNA can be detected in the plasma specimen of 17.9% of Iranian treatment-naive patients with chronic hepatitis B infection. Therefore, designing prospective studies focusing on the detection of cccDNA in these patients would provide more information.     

荧光定量检测HBV cccDNA在慢性乙型肝炎患者中的应用

目的:研究慢性乙型肝炎患者HBV共价闭合环状DNA(cccDNA)在外周血的分布、肝组织中含量及探讨抗病毒治疗对慢性乙型肝炎患者血清HBV cccDNA的影响。方法:随机选取HBV DNA阳性慢性乙型肝炎患者、肝硬化患者、重型肝炎患者共125例。使用实时荧光定量PCR法检测血清中cccDNA。选取36例慢性乙型肝炎患者肝组织和外周血样本,酶切后进行荧光定量PCR检测。60例慢性乙型肝炎患者,按1:1随机分配接受拉米夫定或干扰素治疗,所有患者均治疗24周以上;应用实时荧光定量聚合酶链反应技术检测慢性乙型肝炎患者0周、8周、12周、24周、HBV cccDNA及HBV DNA含量。结果:125例患者中cccDNA阳性率为71.2%,以肝硬化患者阳性率最低。肝硬化患者HBVcccDNA检出阳性率与慢性乙型肝炎患者及重型肝炎患者比较,差异有显著性意义(P0.05)。肝组织中HBV cccDNA与肝组织总HBV DNA及血清HBV DNA存在相关性(P0.05),HBeAg阳性组与阴性组患者比较差异有显著性意义(P0.05)。患者接受抗病毒治疗后血清HBV DNA及HBV cccDNA下降。结论:乙型肝炎肝硬化患者外周血HBV cccDNA阳性率低,慢性乙型肝炎患者中HBeAg(+)组病毒复制较HBeAg(-)组活跃。抗病毒治疗对血清HBV cccDNA有抑制作用;血清HBV cccDNA可作为抗病毒治疗的重要监测指标。     

乙肝患者肝组织中HBVcccDNA与血清中HBVDNA、HBeAg的关系

目的 探讨乙肝患者肝组织中HBVcccDNA与血清中HBVDNA、HBeAg的关系.方法 采用实时荧光定量聚合酶链反应检测乙肝患者肝组织中HBVcccDNA及血清HBVDNA,同时采用ELISA检测其血清中免疫指标HBeAg.结果 30例HBsAg、HBeAg、抗HBc阳性的乙肝患者血清中HBVDNA阳性28例阳性率93.33%,肝组织中HBVcccDNA阳性19例阳性率63.33%,两者比较差异有显著性P＜0.05.30例HBsAg、抗HBe、抗HBc阳性的乙肝患者血清中HBVDNA阳性8例阳性率26.67%,肝组织中HBVcccDNA阳性5例阳性率16.67%,两者比较差异无显著性P＞0.05.10例脂肪肝患者血清HBVDNA阴性,肝组织中未检出HBVcccDNA.结论 (1)血清HBVDNA或HBeAg阳性并不能完全确定肝内一定存在乙肝病毒正在复制.(2)HBeAg阴性抗HBe阳性不能完全确定肝内一定没有乙肝病毒复制.(3)要确定肝内病毒足否存在复制必须通过肝组织HB-VcccDNA检测来证实.     

Requirement of cyclin-dependent kinase function for hepatitis B virus cccDNA synthesis as measured by digital PCR

Introduction and objectivesHBV covalently closed circular (ccc) DNA is the key player in viral persistence and an important predictive biomarker for hepatitis relapse. Precise quantification of intracellular cccDNA is challenging because cccDNA is present in very low levels in hepatocytes, where it also co-exists with a large excess amount of relaxed circular (rc) DNA. We aimed to develop a highly sensitive cccDNA detection method for cccDNA quantification by digital PCR (dPCR).Patients or materials and methodsA standard plasmid containing the whole HBV genome in the closed circular conformation was employed to characterize the performance of dPCR. rcDNA in the growth medium of HBV-producing HepAD38 cells was used as a matrix for cccDNA detection. Intrahepatic cccDNA measurement by dPCR and qPCR was performed to determine the correlation of the analysis results for the two methods.ResultsThe limit of detection (LOD) of the cccDNA dPCR was 1.05 copy/μl, and the linear range of detection was 1.02 × 10⁴ copies/μl, achieving a dynamic detection range of 10⁴-fold. cccDNA measurement using excess rcDNA as the matrix did not reveal false-positive detection, indicating that dPCR was highly specific. In the HepAD38 cells, the cccDNA levels measured by dPCR were highly correlated with those measured by qPCR but had a higher sensitivity. The CDK inhibitor AZD-5438 was found to block intracellular cccDNA synthesis.ConclusionsDpcr greatly improved the sensitivity and specificity of cccDNA detection. Host CDK activities are likely required for cccDNA synthesis. dPCR can potentially be applied for drug screening for effective cccDNA inhibitors.