Forms Of Hepatitis B Virus DNA Research Articles

Approaches to quantifying hepatitis B virus covalently closed circular DNA.

Chronic hepatitis B is a major cause of liver disease worldwide and is currently incurable. Hepatitis B virus (HBV) covalently closed circular (ccc) DNA is a key form of the virus responsible for its persistence and is the transcriptional template for all viral transcripts. The field is focussed on methods to clear HBV cccDNA but this been limited by technical difficulties in its quantification due to: identical sequence to other forms of HBV DNA; low copy number per cell; and high resistance to denaturation by heat, leading to difficulty using polymerase chain reaction or hybridization methods for detection. A number of assays have been developed in order to overcome these hurdles either directly or detecting cccDNA levels indirectly via its transcriptional products. In this review, we summarize the approaches to cccDNA quantification that are currently used, and outline key open questions in the cccDNA biology field which remain to be answered due to the limitations of current methods.

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.

Molecular characteristics of serum hepatitis B virus (HBV) RNA: a novel biomarker for chronic HBV infection

Background and Hypothesis: HBV is a major etiological agent for viral hepatitis and hepatocellular carcinoma. HBV is a DNA virus per se but viral pregenomic RNA (pgRNA) has been recently found in patient blood. The serum pgRNA is hypothesized to function as a novel biomarker for the activity of HBV covalently- closed-circular DNA (cccDNA), which is the intracellular persistent form of HBV DNA and the template for producing viral proteins responsible for the chronic virulence of HBV. However, the molecular characteristics of serum HBV RNA remains elusive. &#x0D; Experimental Design and Project Methods: HBV RNA were extracted from patient’s sera. RT-PCR and 3’ RACE were utilized to amplify the internal sequence and 3’ terminus of HBV pgRNA, respectively. The PCR products were gel purified and cloned into T vector for sequencing. &#x0D; Results: After comparing the sequencing data to the reference HBV sequence, we found that the serum HBV RNA are spliced fragments of the original intracellular full-length pgRNA. The prevalence of the fragmented serum pgRNA was found in greater concentrations in untreated patients, and the ratios of different spliced forms of serum HBV RNA vary during the course of antiviral treatment. &#x0D; Conclusion and Potential Impact: Our study demonstrated that the serum HBV RNA are spliced/truncated forms of pgRNA, indicating that the RNA-containing virion is noninfectious. The characterization of serum HBV RNA sequence provides important insights into the assay design for serum HBV RNA detection. Future study will focus on the mechanism underlying the selective egress of spliced pgRNA-containing virus particles.

Systemic diseases and biotherapies: Understanding, evaluating, and preventing the risk of hepatitis B reactivation

Hepatitis B virus (HBV) reactivation can occur in chronic carriers of the HBV surface antigen (HBsAg) and constitutes a well-known complication of immunosuppressive therapy. HBV reactivation has also been reported after contact with the HBV. The increasing use of biological agents (TNFα antagonists, rituximab, abatacept, and tocilizumab) to treat systemic diseases has resulted in numerous publications about the risk of HBV reactivation. The relevant scientific societies have issued recommendations designed to prevent HBV reactivation. The main measures consist of screening for markers indicating chronic HBV infection (HBsAg) or HBV infection in the distant past (antibodies to the HBV core antigen) before initiating biological therapies, vaccinating marker-negative patients, and considering close follow-up or antiviral treatment before immunosuppressive treatment initiation or in the event of HBV reactivation. Here, we discuss the pathophysiological mechanisms underlying HBV reactivation during biological treatments, most notably in patients with occult HBV infection or markers for remote HBV infection, whose hepatocyte nuclei may contain a resistance form of HBV DNA known as covalently closed circular DNA (cccDNA). Assessment of the risk of reactivation relies on the HBV status, drugs used, and data from the literature. Finally, we discuss the various recommendations and modalities for HBV vaccination, preemptive treatment, and patient management, according to the level of risk and to the circumstances in which reactivation occurs.

An Occult Hepatitis B-Derived Hepatoma Cell Line Carrying Persistent Nuclear Viral DNA and Permissive for Exogenous Hepatitis B Virus Infection

Occult hepatitis B virus (HBV) infection is defined as persistence of HBV DNA in liver tissues, with or without detectability of HBV DNA in the serum, in individuals with negative serum HBV surface antigen (HBsAg). Despite accumulating evidence suggesting its important clinical roles, the molecular and virological basis of occult hepatitis B remains unclear. In an attempt to establish new hepatoma cell lines, we achieved a new cell line derived from a hepatoma patient with chronic hepatitis C virus (HCV) and occult HBV infection. Characterization of this cell line revealed previously unrecognized properties. Two novel human hepatoma cell lines were established. Hep-Y1 was derived from a male hepatoma patient negative for HCV and HBV infection. Hep-Y2 was derived from a female hepatoma patient suffering from chronic HCV and occult HBV infection. Morphological, cytogenetic and functional studies were performed. Permissiveness to HBV infection was assessed. Both cell lines showed typical hepatocyte-like morphology under phase-contrast and electron microscopy and expressed alpha-fetoprotein, albumin, transferrin, and aldolase B. Cytogenetic analysis revealed extensive chromosomal anomalies. An extrachromosomal form of HBV DNA persisted in the nuclear fraction of Hep-Y2 cells, while no HBsAg was detected in the medium. After treated with 2% dimethyl sulfoxide, both cell lines were permissive for exogenous HBV infection with transient elevation of the replication intermediates in the cytosol with detectable viral antigens by immunoflurescence analysis. In conclusions, we established two new hepatoma cell lines including one from occult HBV infection (Hep-Y2). Both cell lines were permissive for HBV infection. Additionally, Hep-Y2 cells carried persistent extrachromosomal HBV DNA in the nuclei. This cell line could serve as a useful tool to establish the molecular and virological basis of occult HBV infection.

Quantification of hepatitis B virus covalently closed circular DNA in patients with hepatocellular carcinoma

This study aimed to measure the intrahepatic total hepatitis B virus (HBV) DNA and covalently closed circular DNA (cccDNA) levels in tumor and non-tumor tissues in hepatocellular carcinoma (HCC) patients. Intrahepatic total HBV DNA and cccDNA were measured in 25 HCC patients (21 hepatitis B surface antigen [HBsAg]-positive and 4 HBsAg-negative) by the Invader assay. A low level of intrahepatic HBV DNA was detectable in all HBsAg-negative patients. For HBsAg-positive patients, the intrahepatic total HBV DNA levels in the tumor and non-tumor tissues were comparable (P=0.903). However, the tumor tissues had significantly higher levels of cccDNA (0.35 vs. 0.16 copies/cell, P=0.030) and higher proportion of intrahepatic HBV DNA in the form of cccDNA (100% vs. 84%, P=0.004) than the non-tumor tissues. Seventeen out of 21 (81%) tumor tissues had intrahepatic HBV DNA solely in cccDNA form. Analysis of HBV mRNA expression indicated that HBV replication appeared to be lower in the tumor tissues than the non-tumor tissues. Compared to the non-tumor tissues, the levels of HBV replication in the tumor tissues appeared to be lower, and cccDNA was the predominant form of HBV DNA in the tumor tissues.

One-Year Entecavir or Lamivudine Therapy Results in Reduction of Hepatitis B Virus Intrahepatic Covalently Closed Circular DNA Levels

Entecavir and lamivudine are potent nucleoside analogues that can suppress hepatitis B virus (HBV) replication. However, the effects of these two antiviral agents on intrahepatic total HBV DNA and covalently closed circular DNA (cccDNA) are not known. In this study, we aimed to assess the effect of 48 weeks of entecavir/lamivudine therapy on intrahepatic total HBV DNA and cccDNA levels. Forty chronic hepatitis B patients, participating in two Phase III entecavir trials at our centre, were randomized to receive 48 weeks of either 0.5 mg once daily of entecavir (n = 21) or 100 mg once daily of lamivudine (n = 19). Their serological, virological and biochemical responses, as well as intrahepatic HBV DNA levels were monitored. There was no significant difference between entecavir and lamivudine therapy in terms of post-treatment serological, virological and biochemical responses. Both nucleoside analogues reduced serum viral load, intrahepatic total HBV DNA, and cccDNA by about 4.8 logs, 2 logs, and 1 log respectively. An increase in the proportion of intrahepatic HBV DNA in the form of cccDNA was seen after 48 weeks of therapy. In conclusion, both entecavir and lamivudine can successfully reduce intrahepatic HBV DNA and cccDNA. CccDNA becomes the dominant form of HBV DNA during viral suppression and is possibly responsible for viral rebound after short-term antiviral therapy.

Role of Hepatitis B Virus in Hepatocarcinogenesis in Alcoholics

Background: The pathogenesis of hepatocellular carcinoma (HCC) in alcoholics is not understood well. Occult hepatitis B virus (HBV) infection may have some roles.Methods: We compared the clinical features of HCC in patients with and without a history of excessive alcohol consumption. For hepatitis B surface antigen (HBsAg)‐negative, anti–hepatitis C virus (HCV)‐negative (NBNC) patients, HBV DNA in the tumor was detected by polymerase chain reaction (PCR)‐Southern blotting. Integrated and episomal forms of HBV DNA were detected using agarose gel electrophoresis after nested PCR and were quantified by real‐time PCR.Results: For patients who were anti–HCV positive in serum, the age at diagnosis of HCC was lower for alcoholics than nonalcoholics. For serum HBsAg‐positive patients and those who were NBNC, the age at diagnosis of HCC was the same for alcoholics and nonalcoholics. The numbers of tumors and of patients with vascular invasion did not differ between alcoholics and nonalcoholics in any subset. In three NBNC‐HCC patients with a history of alcohol abuse, HBV DNA was detected in the tumor. Integrated HBV DNA was detected in one patient, and the episomal form of HBV DNA was detected in two patients.Conclusion: HBV may be found in episomal and integrated forms in the chromosomal DNA of the livers of habitual drinkers who are negative for HBsAg and anti‐HCV and may have a direct role in hepatocarcinogenesis.

Comparison of Anti-Hepatitis B Virus Activities of Lamivudine and Clevudine by a Quantitative Assay

In this study, we used a quantitative assay to measure the concentration-dependent effects of antivirals on extracellular hepatitis B virus (HBV) DNA as well as on different cytoplasmic and nuclear forms of HBV DNA that participate in HBV replication. HBV recombinant baculovirus, which efficiently delivers the HBV genome to HepG2 cells, was used for this study because (i) antivirals can be administered prior to initiation of HBV infection or after HBV infection and (ii) sufficiently high HBV replication levels are achieved that HBV covalently closed circular (CCC) DNA can be easily detected and individual HBV DNA species can be quantitatively analyzed separately from total HBV DNA. The results showed that the levels of HBV replicative intermediate and extracellular DNA decreased in a concentration-dependent fashion following antiviral treatment. The 50% effective concentration (EC(50)) and EC(90) values and the Hill slopes differed for the different HBV DNA species analyzed. The data clearly indicated that (i) nuclear HBV DNAs are more resistant to antiviral therapy than cytoplasmic or extracellular HBV DNAs and (ii) nuclear HBV CCC DNA is more resistant than the nuclear relaxed circular form. This report presents the first in vitro comparison of the effects of two antivirals administered prior to initiation of HBV infection and the first thorough in vitro quantitative study of concentration-dependent antiviral effects on HBV CCC DNA.

Rebound of hepatitis B virus replication in HepG2 cells after cessation of antiviral treatment.

Treatment of patients with lamivudine (3TC) results in loss of detectable levels of hepatitis B virus (HBV) DNA from serum; however, the relapse rate, with regard to both reappearance of virus in the bloodstream and hepatic inflammation, is high when therapy is terminated. Although the rebound observed in patients has also been seen in animal hepadnavirus models, rebound has not been analyzed in an in vitro cell culture system. In this study, we used the HBV recombinant baculovirus/HepG2 system to measure the time course of antiviral agent-mediated loss of HBV replication as well as the time course and magnitude of HBV production after release from antiviral treatment. Because of the sensitivity of the system, it was possible to measure secreted virions, intracellular replicative intermediates, and nuclear non-protein-bound HBV DNA and separately analyze individual species of DNA, such as single-stranded HBV DNA compared to the double-stranded form and relaxed circular compared to covalently closed circular HBV DNA. We first determined that HBV replication in the HBV recombinant baculovirus/HepG2 system could proceed for at least 35 days, with a 30-day plateau level of replication, making it possible to study antiviral agent-mediated loss of HBV followed by rebound after cessation of drug treatment. All HBV DNA species decreased in a time-dependent fashion following antiviral treatment, but the magnitude of decline differed for each HBV DNA species, with the covalently closed circular form of HBV DNA being the most resistant to drug therapy. When drug treatment ceased, HBV DNA species reappeared with a pattern that recapitulated the initiation of replication, but with a different time course.

Use of the hepatitis B virus recombinant baculovirus-HepG2 system to study the effects of (-)-beta-2',3'-dideoxy-3'-thiacytidine on replication of hepatitis B virus and accumulation of covalently closed circular DNA.

(-)-Beta-2',3'-Dideoxy-3'-thiacytidine (lamivudine [3TC]) is a nucleoside analog which effectively interferes with the replication of hepatitis B virus (HBV) DNA in vitro and in vivo. We have investigated the antiviral properties of 3TC in vitro in HepG2 cells infected with recombinant HBV baculovirus. Different types of information can be obtained with the HBV baculovirus-HepG2 system because (i) experiments can be carried out at various levels of HBV replication including levels significantly higher than those that can be obtained from conventional HBV-expressing cell lines, (ii) cultures can be manipulated and/or treated prior to or during the initiation of HBV expression, and (iii) high levels of HBV replication allow the rapid detection of HBV products including covalently closed circular (CCC) HBV DNA from low numbers of HepG2 cells. The treatment of HBV baculovirus-infected HepG2 cells with 3TC resulted in an inhibition of HBV replication, evidenced by reductions in the levels of both extracellular HBV DNA and intracellular replicative intermediates. The effect of 3TC on HBV replication was both dose and time dependent, and the reductions in extracellular HBV DNA that we observed agreed well with the previously reported efficacy of 3TC in vitro. As expected, levels of HBV transcripts and extracellular hepatitis B surface antigen and e antigen were not affected by 3TC. Importantly, the HBV baculovirus-HepG2 system made it possible to observe for the first time that CCC HBV DNA levels are lower in cells treated with 3TC than in control cells. We also observed that the treatment of HepG2 cells prior to HBV baculovirus infection resulted in a slight increase in the efficacy of 3TC compared to treatments starting 24 h postinfection. The treatment of HepG2 cells with the highest concentration of 3TC tested in this study (2 microM) prior to the initiation of HBV replication markedly inhibited the accumulation of CCC DNA, whereas treatment with the same concentration of 3TC at a time when CCC HBV DNA pools were established within the cells was considerably less effective. In addition, our results suggest that in HepG2 cells, non-protein-associated relaxed circular HBV DNA and particularly CCC HBV DNA are considerably more resistant to 3TC treatment than other forms of HBV DNA, including replicative intermediates and extracellular DNA. We conclude from these studies that the HBV baculovirus-HepG2 system has specific advantages for drug studies and can be used to complement other in vitro model systems currently used for testing antiviral compounds.

G1 phase dependent nuclear localization of relaxed-circular hepatitis B virus DNA and aphidicolin-induced accumulation of covalently closed circular DNA.

During chronic hepatitis B virus (HBV) infection, virus persistence relies on the maintenance of a pool of covalently closed circular DNA (cccDNA) in the nuclei of infected hepatocytes. To achieve this, HBV DNA has to be transported from the cytoplasm to the nucleus. By carrying out subcellular fractionation experiment, both of the relaxed-circular (RC) and single-stranded (SS) HBV DNA were found in the cytoplasm whereas only RC form could be detected in the nucleus of a hepatoblastoma cell line (HepG2) stably producing HBV. This fraction of nuclear RC viral DNA was clearly demonstrated in the G1 but not S phase of synchronized HepG2 cells. Conversely, the relative amount of cytoplasmic RC viral DNA in the S phase was larger than that in the G1 phase. Although no cccDNA could be detected in HepG2 cells without synchronization, an increasing amount of cccDNA in the nucleus was demonstrated after prolonged incubation of the cells in aphidicolin. Finally, by undertaking in situ hybridization using a probe specific to plus-strand HBV DNA, nuclear viral DNA was detected predominantly in the G1 phase of HepG2 cells. In summary, the results indicated that only RC but not SS form of HBV DNA was localized to the nuclei of HepG2 cells. The nuclear localization occurred preferentially in the G1 but not S phase and prolonged treatment with aphidicolin resulted in accumulation of nuclear cccDNA.

Interferon alfa therapy in patients with chronic hepatitis B virus infection: Effects on hepatitis B virus DNA in the liver

Pretrial and posttrial liver biopsy samples from 124 adult patients who participated in two randomized, controlled trials of interferon alfa therapy for chronic hepatitis B virus (HBV) infection were analyzed to determine the effects of interferon on the replication of HBV in the liver. Replicative forms of HBV DNA were detected in the pretrial biopsy samples from all and posttrial biopsy samples from 74% treated patients and 86% controls. Replicative forms of HBV DNA were detected in the posttrial biopsy samples from all patients who remained positive for hepatitis B e antigen and HBV DNA in the serum, in 77% treated patients and 80% controls who cleared HBV DNA in the serum but who remained positive for hepatitis B e antigen, but in only 19% treated patients and 40% controls who cleared HBV DNA as well as hepatitis B e antigen in the serum. Serum alanine aminotransferase levels were significantly lower in patients whose posttrial biopsies did not contain replicative forms of HBV DNA. In summary, we demonstrated that in most patients with chronic HBV infection treated with interferon alfa, serological response was associated with the disappearance of replicative forms of HBV DNA in the liver.

Different forms of hepatitis B virus DNA and expression of HBV antigens in peripheral blood mononuclear cells in chronic hepatitis B

The presence of both hepatitis B virus (HBV) DNA and HBV antigens (HBsAg, HBeAg) was assessed in peripheral blood mononuclear cells (PBMC) from 32 patients chronically infected with HBV. Three different molecular forms of HBV DNA were observed: free monomers (5), high-molecular-weight free concatemers (11), and integrated HBV DNA (9). The HBV DNA patterns in the PBMC were different from those found in liver and did not correlate with any specific profile of serum HBV markers. When the same PBMC were assayed for HBsAg, 22 of the 25 HBV DNA positive samples, but only three of the seven HBV DNA negative samples, were positive. By contrast, none of the PBMC samples from five healthy HBV vaccine recipients gave any positive signal in the HBV DNA or HBsAg assays. In some patients, T and B cells, monocytes, and polymorphonuclear (PMN) cells were assayed separately, showing that the DNA pattern was similar for these different leucocytes subsets and ruling out the possibility that these patterns might reflect PMN cell contamination. Thus, in chronic HBV infection, 87.5% (28/32) of patients were found to contain at least one HBV marker in their PBMC, and a strong correlation was found between the presence of HBV DNA and viral antigens, suggesting a specific expression of HBV encoded proteins.

Hepatitis B virus DNA patterns in the liver of children with chronic hepatitis B

The pattern of hepatitis B virus DNA (HBV-DNA) expression were studied in 2 sequential liver biopsies from 26 children (18 treated with interferon and 8 controls) with chronic hepatitis B. In the basal biopsy replicative forms of HBV-DNA were detected in all of the samples and integrated viral DNA was present in 1 case. At the end of the study, 8 children had lost serum HBV-DNA although 2 of the children were still HBeAg positive. (Six had been treated with interferon.) In all of the cases, HBV-DNA was not detectable in the final biopsy. For the rest of the patients, HBV-DNA was positive in serum and all of them had replicative forms of HBV-DNA in the second liver sample. None of the patients lost hepatitis B surface antigen (HBsAg). Peripheral blood mononuclear cells (PBMC) from these patients were studied. HBV-DNA was not found in the PBMC of the 8 children without serum HBV-DNA, and HBV-DNA was detected in the PBMC of 5/12 patients with serum HBV-DNA. In conclusion, HBV-DNA disappeared from the biopsies of children who lost circulating HBV-DNA, although some of the patients were still HBeAg positive. This result implies that the detection of HBV-DNA in liver is important in order to assess the efficacy of the antiviral therapy. On the other hand, HBsAG remained positive in all children at the end of the study although HBV-DNA was not detected in serum, liver, and PBMC by the conventional hybridization techniques.

State of hepatitis B virus DNA in peripheral blood mononuclear cells from persistently infected individuals: correlation with e antigen and viral DNA in the serum as well as with the activity of liver disease.

Peripheral blood mononuclear cells (PBMC) were harvested from 76 asymptomatic carriers of hepatitis B virus (HBV) and 100 patients with type B chronic liver disease. DNA was extracted from cells and tested for the binding with radiolabeled HBV DNA probe by Southern blot hybridization technique. Among 34 asymptomatic carriers who had hepatitis B e antigen (HBeAg) and HBV DNA in the serum, HBV DNA was detected in 29 (85%) PBMC. In remarkable contrast, of the remaining 42 carriers seronegative for HBeAg, only 1 (2%) had HBV DNA in the serum, and none exhibited HBV DNA in PBMC. Among 32 patients seropositive for HBeAg, HBV DNA was detected in 28 (88%) sera, and in 24 (75%) PBMC. Of 68 patients seronegative for HBeAg, HBV DNA was found in 10 (15%) sera, and in 7 (10%) PBMC. Among 62 cases with HBV DNA in PBMC, only 1 had it integrated into the host's DNA. The remaining 61 had free HBV DNA in PBMC. Only 8 of them possessed replicative intermediate forms of HBV DNA, with molecular sizes less than 2.0 kilobases as observed in liver infected with HBV, and they all were patients with chronic active hepatitis. HBV DNA was not detectable in PBMC from 172 controls comprising 44 healthy individuals and 128 patients with non-A, non-B hepatitis. Based on these results, the state of HBV DNA in PBMC reflects the phase of HBV infection with HBeAg in the serum, and a high activity of hepatitis accompanied by active HBV replication.

Biochemical Detection of Hepatitis B Virus Constituents

Publisher Summary This chapter discusses biochemical methods for the detection of hepatitis B virus (HBV) constituents. Hepatitis B virus is the smallest of the DNA viruses that infect man. Only one other animal DNA virus that infects pigs is smaller. The four viruses—HBV, woodchuck hepatitis virus, ground squirrel hepatitis virus, and duck hepatitis virus—form a distinct group of reverse transcribing viruses that show strong similarities in virion size, in the length of the genome, and in other properties. Two biochemical techniques are applicable to the detection of HBV DNA: dot hybridization that provides a semiquantitative test for its presence or absence, and southern blot analysis, by means of which HBV DNA sequences are located in nucleic acid fragments that are separated according to electrophoretic mobility. The principal tool for studying the molecular forms of HBV DNA is the southern blot technique. DNA forms are separated according to size and secondary and tertiary structure by means of gel electrophoresis and are transferred from the gel to a membrane either by blotting or by electrophoresis.

Status of hepatitis B virus DNA in hepatocellular carcinoma: a study based on paired tumor and nontumor liver tissues.

To investigate the hepatitis B virus (HBV) DNA status in the liver when hepatocellular carcinoma (HCC) has developed, 35 paired nontumorous and tumorous liver tissues from 27 hepatitis B surface antigen (HBsAg)-seropositive and 8 HBsAg-negative patients with HCC were studied by Southern blot analysis. The hybridization patterns of HBV DNA were different in the nontumor and tumor parts in 26 (96.3%) of the 27 HBsAg-positive patients. In the nontumor parts, integration of HBV DNA into the host genome was significantly less when compared to the tumor parts (15/27 vs. 25/27, P less than 0.05), whereas free replicative viral forms were significantly more frequent (17/27 vs. 7/27). The integrated HBV DNA in the nontumor parts showed discrete band patterns in the majority of cases (13/15). Hepatitis B e antigen (HBeAg) was significantly associated with the expression of free replicative forms of HBV DNA in the tumor tissues. An integrated HBV DNA sequence was detected in the tumor part of one HBsAg-negative patient, but not in her nontumor counterpart. Our observation that discrete integrated HBV DNAs are present in the nontumor part, representing subclinical clonal expansion that precedes the development of HCC, suggests the risk of future new tumor growth from these cell clones.

Detection of hepatitis B virus DNA in human serum by dot hybridization using a biotin-labelled probe.

A dot blot hybridization technique utilizing a biotin-labelled recombinant DNA probe was used to examine hepatitis B virus (HBV) DNA in serum. The lowest amount of HBV DNA in serum detectable by the color development of an avidin-biotin alkaline phosphatase complex was 40 picogram per 50 microliter. Validity of this method was confirmed by autoradiography using 32P-labelled and 3H-labelled HBV DNA probes. HBV DNA was found in 100% (34/34) of the HBsAg-positive and in 73.5% (25/34) of the HBsAg-negative subjects. In contrast, all nine cases showing negativity in HBV DNA were also HBsAg-negative. Correlation of HBe antigen/antibody with HBV DNA was investigated in 19 sera of which HBsAg was negative but anti-HBc-positive. Of 13 sera with anti-HBe 10 (76.9%) cases revealed HBV DNA positivity, while four (66.7%) of six sera without anti-HBe were positive in HBV DNA.In conclusion, serum dot hybridization assay utilizing a biotinylated probe proved useful in the detection of a free form of HBV DNA regardless of the presence of HBsAg and irrespective of HBeAg/anti-HBe status. Moreover, it is emphasized that practical advantages in speed, reproducibility, and safety have made this alternative even more attractive than autoradiography using radioisotope-labelled probes.