Detection Of cccDNA Research Articles

A Chromogenic In Situ Hybridization (CISH) Assay for Detection of HBV RNA, DNA, and cccDNA in Liver Tissue.

Hepatitis B virus (HBV) developed highly intricates mechanisms exploiting host resources for its multiplication within a constrained genetic coding capacity. With the aid of a series of classical analytical methods such as ultrafiltration, and Southern and Northern blots, a general framework of HBV life cycle has been established. However, this picture still lacks many key histological contexts which involves pathophysiological changes of hepatocytes, non-parenchymal cells, infiltrated leukocytes, and associated extracellular matrix. Here, we describe a CISH protocol modified from the ViewRNA assay that allows direct visualization of HBV RNA, DNA, and cccDNA in liver tissue of chronic hepatitis B patients. By coupling it with immunohistochemistry and other histological stains, much richer information regarding the HBV-induced pathological changes can be harvested.

A Fluorescent In Situ Hybridization (FISH) Assay for Detection of HBV RNA, DNA, and cccDNA in Cell Culture.

Hepatitis B virus (HBV) is undoubtedly a master in exploiting host resources while evading host defense for its multiplication within a constrained genetic coding capacity. To further unravel these cunning strategies, a clear picture of virus-host interaction with key subcellular and molecular contexts is needed. Here, we describe a FISH protocol modified from the ViewRNA assay that allows direct visualization of HBV RNA, DNA, and cccDNA in cell culture models (e.g., HepAD38, HepG2-NTCP). It can be coupled with immunofluorescence staining of viral or host proteins or other fluorescent tagging systems which could illuminate numerous aspects of virus-host interactions.

Rapid and Reliable Protein-Free HBV DNA Extraction and Sensitive Branched DNA Southern Blot Assay.

HBV covalently closed circular DNA (cccDNA) plays an important role in the persistence of hepatitis B virus (HBV) infection by serving as the template for transcription of viral RNAs. To cure HBV infection, it is expected that cccDNA needs either to be eliminated or silenced. Hence, precise cccDNA quantification is essential. Sample preparation is crucial to specifically detect cccDNA. Southern blot is regarded as the "gold standard" for specific cccDNA detection but lacks sensitivity. Here, we describe a rapid and reliable modified kit-based, HBV protein-free DNA extraction method as well as a novel enhanced sensitivity Southern blot that uses branched DNA technology to detect HBV DNA in cell culture and liver tissue samples. It is useful for both HBV molecular biology and antiviral research.

Research progress on detection methods for hepatitis B virus covalently closed circular DNA.

Hepatitis B virus (HBV) infection remains a serious global public health problem, and HBV covalently closed circular DNA (cccDNA) in the nucleus of infected cells cannot be eliminated by current treatments and is a major factor in the persistence and recurrence of hepatitis B. Efficient and scientific detection methods are important for clinical monitoring of cccDNA and targeted drug development. Western blotting is the gold standard for the quantitative detection of cccDNA, but it is time-consuming and complex. In recent years, new detection technologies have been continuously updated. There are new developments and breakthroughs in both next-generation polymerase chain reaction (PCR) and non-PCR methods such as in situ hybridization. Some HBV-related markers (such as hepatitis Bcore-relatedantigen) have also been shown to be closely related to cccDNA, and they can be used as surrogate markers to indirectly reflect cccDNA content. In this paper, the main detection methods of cccDNA and their improvements are reviewed, the advantages and limitations of these methods are analysed and summarized, and future development directions are proposed.

The impact of the covalently closed circular DNA level on recurrence of hepatocellular carcinoma after initial hepatectomy: an analysis of patients with resolved hepatitis B virus infection.

We assessed whether or not covalently closed circular DNA (cccDNA) levels in the background liver influence the recurrence of hepatocellular carcinoma (HCC) in patients with resolved hepatitis B virus (HBV) infection. Among 425 patients who underwent initial hepatectomy for HCC between 2010 and 2018, a retrospective review was performed in 44 with resolved HBV infection. The clinicopathologic characteristics were analyzed for correlation with tumor recurrence. The HBV cccDNA levels were tested via a droplet digital polymerase chain reaction assay. HBV cccDNA was detected in 27 of 44 patients (61%), and the median level was 1.0 copies/1000 ng (range, 0-931.3 copies/1000 ng). Anti-HBc ≥8.9 S/CO was associated with cccDNA detection (odds ratio, 11.08; 95% confidence interval [95% CI], 2.48-49.46; P = 0.002). Twenty-eight patients (64%) developed HCC recurrence after hepatectomy. The overall 3- and 5-year recurrence-free survival rates were 45.7% and 34.3%, respectively.19 HBV cccDNA levels was not significantly associated with HCC recurrence, while the presence of multiple tumors was an independent risk fact or (hazard ratio, 6.53; 95% CI, 2.48-17.19; P < 0.001. HBV cccDNA levels did not influence HCC recurrence after hepatectomy. Anti-HBc levels may be used as a surrogate marker for cccDNA.

CRISPR/Cas13-assisted hepatitis B virus covalently closed circular DNA detection

Background and aimsThe formation of an intranuclear pool of covalently closed circular DNA (cccDNA) in the liver is the main cause of persistent hepatitis B virus (HBV) infection. Here, we established highly sensitive and specific methods to detect cccDNA based on CRISPR-Cas13a technology.MethodsWe used plasmid-safe ATP-dependent DNase (PSAD) enzymes and HindIII to digest loose circle rcDNA and double-stranded linear DNA, amplify specific HBV cccDNA fragments by rolling circle amplification (RCA) and PCR, and detect the target gene using CRISPR-Cas13a technology. The CRISPR-Cas13a-based assay for the detection of cccDNA was further clinically validated using HBV-related liver tissues, plasma, whole blood and peripheral blood mononuclear cells (PBMCs).ResultsBased on the sample pretreatment step, the amplification step and the detection step, we established a new CRISPR-Cas13a-based assay for the detection of cccDNA. After the amplification of RCA and PCR, 1 copy/μl HBV cccDNA could be detected by CRISPR/Cas13-assisted fluorescence readout. We used ddPCR, qPCR, RCA-qPCR, PCR-CRISPR and RCA-PCR-CRISPR methods to detect 20, 4, 18, 14 and 29 positive samples in liver tissue samples from 40 HBV-related patients, respectively. HBV cccDNA was almost completely undetected in the 20 blood samples of HBV patients (including plasma, whole blood and PBMCs) by the above 5 methods.ConclusionsWe developed a novel CRISPR-based assay for the highly sensitive and specific detection of HBV cccDNA, presenting a promising alternative for accurate detection of HBV infection, antiviral therapy evaluation and treatment guidance.

Droplet digital PCR assay provides intrahepatic HBV\xa0cccDNA quantification tool for clinical application

The persistence of covalently closed circular DNA (cccDNA) poses a major obstacle to curing chronic hepatitis B (CHB). Here, we used droplet digital PCR (ddPCR) for cccDNA quantitation. The cccDNA-specific ddPCR showed high accuracy with the dynamic range of cccDNA detection from 101 to 105 copies/assay. The ddPCR had higher sensitivity, specificity and precisely than qPCR. The results of ddPCR correlated closely with serum HB core-related antigen and HB surface antigen (HBsAg) in 24 HBV-infected human-liver-chimeric mice (PXB-mice). We demonstrated that in 2 PXB-mice after entecavir treatment, the total cccDNA content did not change during liver repopulation, although the cccDNA content per hepatocyte was reduced after the treatment. In the 6 patients with HBV-related hepatocellular carcinoma, ddPCR detected cccDNA in both tumor and non-tumor tissues. In 13 HBeAg-negative CHB patients with pegylated interferon alpha-2a, cccDNA contents from paired biopsies were more significantly reduced in virological response (VR) than in non-VR at week 48 (p = 0.0051). Interestingly, cccDNA levels were the lowest in VR with HBsAg clearance but remained detectable after the treatment. Collectively, ddPCR revealed that cccDNA content is stable during hepatocyte proliferation and persists at quantifiable levels, even after serum HBsAg clearance.

Establishment of a droplet digital PCR method for the detection of hepatitis B virus covalently closed circular DNA

Objective To establish a droplet digital PCR (ddPCR) method for detecting hepatitis B virus (HBV) covalently closed circular DNA (cccDNA). Methods HBV cccDNA standard substance was constructed, and HBV cccDNA primers and probes were designed based on the structural differences between HBV cccDNA and relaxed circular DNA (rcDNA). HBV plasmid was amplified to obtain HBV cccDNA standard substance, and a ddPCR detection method was established with the standard substance after gradient dilution as the template for HBV cccDNA detection; the limit of detection and repeatability of this method were analyzed. Liver tissue samples were collected from 20 patients who attended Beijing YouAn Hospital, Capital Medical University, from June 2017 to October 2020, all of whom were diagnosed with HBV infection, and DNA of the samples was extracted and digested with plasmid-safe ATP-dependent DNA enzyme to obtain HBV cccDNA template; the ddPCR detection method was evaluated in clinical samples and was compared with the quantitative real-time PCR (qPCR) detection method. The chi-square test was used for comparison of categorical data between the two groups. Results The HBV cccDNA detection method based on ddPCR was established, which accurately detected HBV cccDNA in standard substance after gradient dilution, with a limit of detection of 1 copy/μl, and the coefficients of variation of 1×103, 1×102, and 1×101 copies/μl standard substances were 4.41%, 3.98%, and 5.09%, respectively. HBV cccDNA was detected in the samples of 20 patients with HBV infection; the ddPCR detection method detected HBV cccDNA in 17 patients, with a positive rate of 85%, while the qPCR detection method detected HBV cccDNA in 11 patients, with a positive rate of 55%, and there was a significant difference between the two methods (χ2=4.286, P=0.038). Conclusion The established ddPCR method for detecting HBV cccDNA has a low limit of detection and good repeatability, which provides an effective tool for further clinical detection.

A Sensitive and Specific PCR-based Assay to Quantify Hepatitis B Virus Covalently Closed Circular (ccc) DNA while Preserving Cellular DNA.

Hepatitis B virus (HBV) is the major cause of liver diseases and liver cancer worldwide. After infecting hepatocytes, the virus establishes a stable episome (covalently closed circular DNA, or cccDNA) that serves as the template for all viral transcripts. Specific and accurate quantification of cccDNA is difficult because infected cells contain abundant replicative intermediates of HBV DNA that share overlapping sequences but arranged in slightly different forms. HBV cccDNA can be detected by Southern blot or qPCR methods which involve enzymatic digestion. These assays are laborious, have limited sensitivity, or require degradation of cellular DNA (which precludes simple normalization). The method described in this protocol, cccDNA inversion quantitative (cinq)PCR, instead uses a series of restriction enzyme-mediated hydrolysis and ligation reactions that convert cccDNA into an inverted linear amplicon, which is not amplified or detected from other forms of HBV DNA. Importantly, cellular DNA remains quantifiable during sample preparation, allowing normalization and markedly improving precision. Further, a second linear fragment (derived from enzymatic digestion of a separate region of the HBV DNA genome and is present in all forms of HBV DNA) can be used to simultaneously quantify total HBV levels. Graphic abstract: Selective detection of HBV cccDNA and total HBV DNA using cinqPCR (Reproduced from Tu et al., 2020a ).

Quantatitative assays for covalently closed circular DNA of hepatitis B virus

<p indent=0mm>Chronic infection of hepatitis B virus (HBV) is still one of the public health concerns affecting human health. Although the prophylactic HBV vaccines was introduced over three decades ago, the total number of chronic hepatitis B (CHB) infection still accounts for 3.5% (257 million) of the world’s population, with approximately 800000 annual deaths related to the viral infection. Currently, drugs including PEG-IFN and NAs are widely used in the treatment of HBV infection with certain degree of success. It is still difficult to achieve a high proportion of “clinical cure” with existing treatment methods. The presence of covalently closed circular DNA (cccDNA) in the infected hepatocytes is the underlying mechanism for the long-term HBV persistence and is also essential for off-therapy viral relapse. The current treatments have no or limited effects on the suppression of intrahepatic cccDNA. The availability for a reliable cccDNA quantification assay would facilitate the developmentof anti-viral drugs targeting cccDNA-targeting. In this review, we summarized the advantages and disadvantages of the existing methods for cccDNA quantitation. Specifically, the extraction method of Hirt DNA combined with Exonuclease V can effectively remove other forms of HBV DNA, and suitable for cccDNA extraction, but it needs to be further optimized. Southern blot is considered as the “gold-standard” method for cccDNA detection, but it is less sensitive and requires cumbersome procedures, making it an unlikely method for high-throughput screening used for drug development or for clinical applications. The PCR-based methods are more convenient and sensitive for cccDNA detection. However, the reliability of PCR-based method requires further optimization and validation. Different PCR-based methods were developed for quantitation of HBV cccDNA. They include selective qPCR, droplet-digital PCR, rolling circle amplification PCR,<italic> in situ</italic> PCR, invader assay, magnetic capture hybridization qPCR, etc. These PCR-based methods are widely used in clinical and laboratory research for quantitation of cccDNA. Efforts need to be made to minimize the interference from other forms of DNAs in the assays. More recently, hepatitis B core related antigen (HBcrAg)was found to be a novel marker of HBV, and its level in serum is highly correlated with intracellular cccDNA level. This biomarker, the HBcrAg level, can be used to indicate the suppression of cccDNA during different clinical antiviral therapies. The correlation needs further analysis with additional data sets. In summary, we present a review on the quantitation assays for HBV cccDNA. Such assays are important for drug developments and clinical applications for improved management of HBV infection related diseases. Performance and characteristics of various methods were compared with the areas of improvements for different assays.

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 1.05copy/μl, and the linear range of detection was 1.02×104copies/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.

Advances in the detection and clearance of hepatitis B virus covalently closed circular DNA (cccDNA)

Chronic hepatitis B (CHB) is a worldwide public health problem and current therapeutic drugs cannot completely eliminate the covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) in the liver cell nucleus. The presence of cccDNA leads to the persistent HBV infection, which is hard to be completely eliminated. At present, the research on cccDNA is getting increasing attention. Only by comprehensively understanding it can we find a better way to cure HBV infection. This article reviewed the structure, regulation, detection and elimination of HBV cccDNA. Key words: HBV; cccDNA; Detection; Elimination

&amp;lt;italic&amp;gt;In vitro&amp;lt;/italic&amp;gt; experimental models for the study of hepatitis B virus cccDNA

Chronic infection of hepatitis B virus (HBV) remains a global public health problem and affects at least 240 million people worldwide. HBV is a small enveloped DNA virus belonging to the Hepadnaviridae family. The virus has a narrow host range, limited to humans and chimpanzees. In the nucleus of infected hepatocytes, the partially double-stranded DNA (rcDNA) HBV genome is converted to covalent closed circular DNA (cccDNA), which serves as the sole template for HBV transcription. HBV cccDNA exists as an episomal minichromosome in the nucleus, with a turnover rate that correlates with the mitosis or death of virus-infected hepatocytes. It is thus regarded as a primary molecular mechanism for viral persistence. In the past decade, significant progress has been made in HBV research and the related antiviral development. Sodium taurocholate co-transporting polypeptide (NTCP) has been identified as a receptor for HBV entry into hepatocytes. It was recently shown that HBV x (HBx), an enigmatic viral protein, achieves its regulatory function by hijacking the cellular DDB1-containing E3 ubiquitin ligase to target the Smc5/6 complex for degradation. Although still controversial, type I interferon has been implicated to either affect the epigenetic control of cccDNA minichromosome, or induce cytidine deaminases-mediated specific degradation of the episomal cccDNA. Of particular note, core protein allosteric modulator (CpAM) has been most widely investigated. These studies represent a new frontier in the development of antivirals against HBV infection. Despite continuing progress in HBV research, a complete cure of HBV infection is still not achievable. Current antiviral therapies with nucleos(t)ide analogs (NUC) and pegylated IFN-α can efficiently inhibit HBV replication, but the treatments fail to eliminate the preexisting cccDNA pool that is responsible for a viral rebound after therapy cessation. Therefore, it is widely held that cccDNA elimination is a prerequisite for complete cure of chronic HBV infection. To this end, it is essential to understand the mechanisms involved in cccDNA metabolism and transcriptional regulation. Studies of HBV cccDNA have often been hampered by the low copy number of cccDNA per cell and the lack of convenient techniques with high sensitivity and specificity for cccDNA detection. Development of appropriate experimental models is thus fundamental for HBV cccDNA studies. Herein, we reviewed recent advances in cell culture models for the study of HBV cccDNA, including models of HBV de novo infection and cccDNA biogenesis, cccDNA formation (rcDNA repair-related molecular events), HBV replicon-based transfection models to study established cccDNA, and the surrogate model of cccDNA produced from site-specific DNA recombination. Animal models to study HBV cccDNA will be further discussed by other authors in this issue. We hope this review may help clinicians and researchers who are interested in the study of cccDNA and antiviral development against HBV.

A quantitative method for hepatitis B virus covalently closed circular DNA enables distinguishing direct acting antivirals from cytotoxic agents

Studying the biogenesis of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and developing anti-HBV agents require analytical methods to quantify viral DNA levels inside host cells. The well-accepted Southern blotting method is only semi-quantitative, while the other widely used methods (based on qPCR) have been questioned as to their fidelity for cccDNA quantification. In addition, Southern blotting and qPCR results barely reflect the number of host cells present in an analytical sample. We here developed new techniques that substantially improve cccDNA detection and quantification, including a sample pretreatment method that i) exploits high temperature and exonuclease V (an ATP-dependent, bidirectional exonuclease) digestion to effectively increase the amplification efficiency of HBV cccDNA by removing rcDNA and denaturing the cccDNA template, and ii) a method that splits cell samples and uses separate extraction technologies to facilitate “host normalization” based on host genomic DNA signals. Our study introduces new analytical techniques that should be useful for the basic biology and translational studies of HBV.

Characterization of the molecular events of covalently closed circular DNA synthesis in de novo Hepatitis B virus infection of human hepatoma cells

Despite the utmost importance of cccDNA in HBV biology, the mechanism by which cccDNA synthesis is regulated is not completely understood. Here we explored HepG2-NTCP cell line and performed a time-course HBV infection experiment (up to 30 days) to follow the conversion of the input viral DNA into cccDNA. We found that a protein-free RC DNA (PF-RC DNA) become detectable as early as 12 h post infection (hpi) prior to the detection of cccDNA, which become evident only at 2–3 dpi. Intriguingly, the PF-RC DNA detected at 12 hpi was abundantly located in the cytoplasm, implicating that the protein-removal from the input viral DNA takes place in the cytoplasm, perhaps inside the nucleocapsid. Notably, during the early time points of HBV infection, the PF-RC DNA accumulated at significantly higher levels and appeared in a peak followed by a plateau at late time points with dramatically lower levels, implicating the presence of two distinct populations of the PF-RC DNA. Importantly, the PF-RC DNA at earlier peak is entecavir (ETV)-resistant, whereas the PF-RC DNA at posterior days is ETV-sensitive. An interpretation is that the PF-RC DNA at earlier peak represents “input viral DNA” derived from HBV inoculum, whereas the PF-RC DNA at late time points represents the de novo product of the viral reverse transcription. The existence of two populations of the PF-RC DNA having a distinct kinetic profile and ETV-sensitivity implicated that intracellular amplification via the viral reverse transcription greatly contributes to the maintenance of cccDNA pool during HBV infection. As such, we concluded that the cccDNA level is stably maintained by continuing replenishment of cccDNA primarily through intracellular amplification in the HepG2-NTCP cell line.

Quantification of Hepatitis B Virus Covalently Closed Circular DNA in Infected Cell Culture Models by Quantitative PCR.

Persistence of the human hepatitis B virus (HBV) requires the maintenance of covalently closed circular (ccc)DNA, the episomal genome reservoir in nuclei of infected hepatocytes. cccDNA elimination is a major aim in future curative therapies currently under development. In cell culture based in vitro studies, both hybridization- and amplification-based assays are currently used for cccDNA quantification. Southern blot, the current gold standard, is time-consuming and not practical for a large number of samples. PCR-based methods show limited specificity when excessive HBV replicative intermediates are present. We have recently developed a real-time quantitative PCR protocol, in which total cellular DNA plus all forms of viral DNA are extracted by silica column. Subsequent incubation with T5 exonuclease efficiently removes cellular DNA and all non-cccDNA forms of viral DNA while cccDNA remains intact and can reliably be quantified by PCR. This method has been used for measuring kinetics of cccDNA accumulation in several in vitro infection models and the effect of antivirals on cccDNA. It allowed detection of cccDNA in non-human cells (primary macaque and swine hepatocytes, etc.) reconstituted with the HBV receptor, human sodium taurocholate cotransporting polypeptide (NTCP). Here we present a detailed protocol of this method, including a work flowchart, schematic diagram and illustrations on how to calculate "cccDNA copies per (infected) cell".

Exonuclease I and III improve the detection efficacy of hepatitis B virus covalently closed circular DNA

BackgroundHepatitis B virus covalently closed circular DNA (HBV cccDNA) is an important biomarker of hepatitis B virus infection. However, the current methods are not specific and sensitive. The present study aimed to develop a specific and sensitive assay method for the quantification of HBV cccDNA. MethodsExonuclease I (Exo I) & Exonuclease III (Exo III) and specific primer probes are used in real-time PCR. The virus particles isolated from peripheral blood mononuclear cells were used as negative control and HBV1.3 recombinant plasmid 3.2 kb circular DNA fragment was used as positive control. The methods of cccDNA detection were evaluated in cell lines, plasmid, animal model, patient serum and liver biopsies. ResultsA linear range of 101–107 copies/assay using specific primers for HBV cccDNA was established. HBV cccDNA were only detected in cell lines, animal model and liver tissue. It cannot be detected in serum samples. Intrahepatic HBV cccDNA level had good correlation with intrahepatic total HBV DNA level (r = 0.765, P < 0.001). ConclusionsThe real-time quantitative PCR is an effective and feasible method for sensitive and specific detection of low copy number of cccDNA. The novel detection method is fast, provides high sensitivity and specificity and can be used in clinical practice.

Quantitation of HBV cccDNA in anti-HBc-positive liver donors by droplet digital PCR: A new tool to detect occult infection

The accurate diagnosis of occult hepatitis B virus (HBV) infection (OBI) requires the demonstration of HBV DNA in liver biopsies of hepatitis B surface antigen-negative individuals. However, in clinical practice a latent OBI is deduced by the finding of the antibody to the hepatitis B core antigen (anti-HBc). We investigated the true prevalence of OBI and the molecular features of intrahepatic HBV in anti-HBc-positive individuals. The livers of 100 transplant donors (median age 68.2 years; 64 males, 36 females) positive for anti-HBc at standard serologic testing, were examined for total HBV DNA by nested-PCR and for the HBV covalently closed circular DNA (HBV cccDNA) with an in-house droplet digital PCR assay (ddPCR) (Linearity: R2 = 0.9998; lower limit of quantitation and detection of 2.4 and 0.8 copies/105 cells, respectively). A total of 52% (52/100) of the individuals studied were found to have OBI. cccDNA was found in 52% (27/52) of the OBI-positive, with a median 13 copies/105 cells (95% CI 5-25). Using an assay specific for anti-HBc of IgG class, the median antibody level was significantly higher in HBV cccDNA-positive than negative donors (17.0 [7.0-39.2] vs. 5.7 [3.6-9.7] cut-off index [COI], respectively, p = 0.007). By multivariate analysis, an anti-HBc IgG value above 4.4 COI was associated with the finding of intrahepatic HBV cccDNA (odds ratio 8.516, p = 0.009); a lower value ruled out its presence with a negative predictive value of 94.6%. With a new in-house ddPCR-based method, intrahepatic HBV cccDNA was detectable in quantifiable levels in about half of the OBI cases examined. The titer of anti-HBc IgG may be a useful surrogate to predict the risk of OBI reactivation in immunosuppressed patients. The covalently closed circular DNA (cccDNA) form of the hepatitis B virus (HBV) sustains the persistence of the virus even decades after resolution of the symptomatic infection (occult HBV infection). In the present study we developed a highly sensitive method based on droplet digital PCR technology for the detection and quantitation of HBV cccDNA in the liver of individuals with occult HBV infection. We observed that the amount of HBV cccDNA may be inferred from the titer in serum of the IgG class antibody to the hepatitis B core antigen. The quantitation of this antibody may represent a surrogate to determine which patients are at the highest risk of HBV reactivation following immunosuppressive therapies.

Detection of HBV Covalently Closed Circular DNA.

Chronic hepatitis B virus (HBV) infection affects approximately 240 million people worldwide and remains a serious public health concern because its complete cure is impossible with current treatments. Covalently closed circular DNA (cccDNA) in the nucleus of infected cells cannot be eliminated by present therapeutics and may result in persistence and relapse. Drug development targeting cccDNA formation and maintenance is hindered by the lack of efficient cccDNA models and reliable cccDNA detection methods. Southern blotting is regarded as the gold standard for quantitative cccDNA detection, but it is complicated and not suitable for high-throughput drug screening, so more sensitive and simple methods, including polymerase chain reaction (PCR)-based methods, Invader assays, in situ hybridization and surrogates, have been developed for cccDNA detection. However, most methods are not reliable enough, and there are no unified standards for these approaches. This review will summarize available methods for cccDNA detection. It is hoped that more robust methods for cccDNA monitoring will be developed and that standard operation procedures for routine cccDNA detection in scientific research and clinical monitoring will be established.

Advances in detection of covalently closed circular DNA of hepatitis B virus

HBV belongs to the hepatotropic DNA virus family and replicates through reverse transcription. One of the most critical steps in the viral life cycle is that HBV rcDNA (relaxed circular DNA) is transferred to the nucleus where it is converted into cccDNA (covalently closed circular DNA). cccDNA serves as a transcription template for mRNAs and pgRNA, which are ready for the production of new virions. Currently, most antiviral agents can not eliminate HBV cccDNA effectively, which is the key reason for persistent virus infection, reactivation of virus after termination of antiviral therapy and drug resistance. In addition, HBV cccDNA has become an effective index for identifying the states of HBV infection and evaluating efficacy of antiviral therapy. Here, we review research progresses in detection and clinical significance of HBV cccDNA. Key words: Hepatitis B virus; Covalently closed circular DNA; Relaxed circular DNA