Hepatitis B Virus DNA Fragments Research Articles

Research progress on the effect of hepatitis B virus DNA integration on antiviral therapy

Hepatitis B virus (HBV) DNA integration occurs during the reverse transcription process of HBV replication, which develops in the early stages of HBV infection and accompanies the entire disease course. The integration of HBV DNA is detrimental to the attainment of clinical cure goals and also raises the risk of developing liver cancer. Theoretically, nucleos(t)ide analogs can reduce the synthesis of new double-stranded linear DNA, but there is no clearance function for hepatocytes that have already integrated HBV. Therefore, patients with serum HBV DNA-negative conversions still have the risk of developing liver cancer. As an immunomodulatory drug, interferon can not only inhibit viral replication but also inhibit or even eliminate existing clonally amplified hepatocytes carrying integrated HBV DNA fragments. However, there are currently few studies on the effects of nucleos(t)ide analogues and interferon therapy on HBV DNA integration. Thus, large-scale clinical studies are urgently needed for further clarification.

Two fragments of HBV DNA integrated into chrX: 11009033 and its genetic regulation in HepG2.2.15.

Hepatitis B virus (HBV) integration into human genome causes hepatocellular carcinoma (HCC). The present study used inverse nested PCR; the full sequence of HBV DNA fragments of the chrX: 111009033 integration site was detected (987bp), containing two fragments of double‑stranded linear DNA with the same orientation (1,744‑1,094 and 1,565‑1,228nt). By reverse transcription‑quantitative PCR, HBV‑cell fusion transcript was observed in HepG2.2.15 cells. The mean copy number of this site in cells with H2O2 treatment (8.73x10‑2±1.65x10‑2 copies/cell) was significantly higher than that in the cells without H2O2 treatment (3.02x10‑2±2.33x10‑2 copies/cell; P<0.0001). The mean levels of P21‑activated kinase3 (PAK3) were 15.67±5.65 copies/cell in HepG2.2.15 cells with H2O2 treatment, significantly higher than in the cells without H2O2 treatment (11.34±4.58 copies/cell, P=0.0076) and in HepG2 cells (5.92±1.54 copies/cell, P<0.0001). Significant difference of PAK3 levels was also found between HepG2.2.15 cells without H2O2 treatment and HepG2 cells (11.34±4.58 vs. 5.92±1.54 copies/cell, P<0.0001). The average copy numbers of the integration site chrX: 111009033 were positively correlated with the average levels of PAK3 (P=0.0013). The overall trend of PAK3 expression was significantly increased in HepG2.2.15 cells with H2O2 treatment compared with that in HepG2.2.15 cells without H2O2 treatment (37.63±8.16 and 31.38±7.94, P=0.008) and HepG2 cells (21.67±7.88, P<0.0001). In summary, the chrX: 11009033 integration site may originate from primary human hepatocytes, occurrence and clonal expansion of which may upregulate PAK3 expression, which may contribute to hepatocarcinogenesis.

Fluorescence microarrays for enzyme-free DNA detection based on web hybrid chain reaction

The diagnosis of pathogenic microorganism infection mainly relies on molecular detection of nucleic acids or proteins, and the nucleic acid detection is the gold standard for the pathogen detection. The viral genome mutates quickly, and the genome of the host cell usually binds to multiple sites in the viral genome, leading to patient infections. Therefore, rapid nucleic acid detection methods with high specificity are essential for controlling the spread of viruses and maintaining human health. In this study, Cy3-labeled DNA probes were obtained based on a convenient and enzyme-free web hybridization chain reaction (wHCR) and used for DNA diagnosis of hepatitis B virus (HBV) in an isothermal platform. In this microarray platform, the target DNA is directly captured on the microarray and identified by the aggregated DNA probes to amplify the fluorescence signal. After fluorescence scanning analysis, the detection limit of HBV DNA fragments on this DNA microarray is 84 fM. In addition, this method not only specifically distinguishes single-base mismatched sequences, but also obtains the quantitative detection of HBV DNA in serum samples. Compared with the enzymatic amplification reaction in which the target nucleic acids are used as the amplification template, this method effectively avoids cross-contamination. Furthermore, compared to enzymatic reactions, the requirements for reaction conditions such as temperature and pH are relatively lenient. This method is user-friendly and suitable for molecular detection of infectious diseases under limited resource conditions.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection.

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by &lt;em&gt;In Vitro&lt;/em&gt; Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Potential use of serum HBV RNA in antiviral therapy for chronic hepatitis B in the era of nucleos(t)ide analogs.

Although the efficacy of nucleos(t)ide analogue (NA) has been confirmed for treatment of chronic hepatitis B, long-term therapy has been recommended due to the high frequency of off-therapy viral DNA rebound and disease relapse. In this review, the RNA virion-like particles of hepatitis B virus (HBV) are integrated into the life cycle of HBV replication, and the potential significance of serum HBV RNA is systematically described. The production of HBV RNA virion-like particles should not be blocked by NA; in this regard, serum HBV RNA is found to be a suitable surrogate marker for the activity of intrahepatic covalently closed circular DNA (cccDNA), particularly among patients receiving NA therapy. Therefore, the concept of virological response is redefined as persistent loss of serum HBV DNA and HBV RNA. In contrast to hepatitis B surface antigen (HBsAg) that can originate from either the cccDNA or the integrated HBV DNA fragment, serum HBV RNA, with pregenomic RNA origination, can only be transcribed from cccDNA. Therefore, the loss of serum HBV RNA would likely be a promising predicator for safe drug discontinuation. The clinical status of consistent loss of serum HBV RNA accompanied with low serum HBsAg levels might be implicated as a "para-functional cure," a status nearly close to the functional cure of chronic hepatitis B, to distinguish the "functional cure" characterized as serum HBsAg loss with or without anti-HBs seroconversion.

Removal of Integrated Hepatitis B Virus DNA Using CRISPR-Cas9.

The presence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and the permanent integration of HBV DNA into the host genome confers the risk of viral reactivation and hepatocellular carcinoma. Nucleoside/nucleotide analogs alone have little or no capacity to eliminate replicative HBV templates consisting of cccDNA or integrated HBV DNA. Recently, CRISPR/Cas9 technology has been widely applied as a promising genome-editing tool, and HBV-specific CRISPR-Cas9 systems were shown to effectively mediate HBV cccDNA disruption. However, the integrated HBV DNA fragments are considered as important pro-oncogenic properties and it serves as an important template for viral replication and expression in stable HBV cell line. In this study, we completely excised a full-length 3,175-bp integrated HBV DNA fragment and disrupted HBV cccDNA in a stable HBV cell line. In HBV-excised cell line, the HBV cccDNA inside cells, supernatant HBV DNA, HBsAg, and HBeAg remained below the negative critical values for more than 10 months. Besides, by whole genome sequencing, we analyzed off-target effects and excluded cell contamination. It is the first time that the HBV infection has been fully eradicated in a stable HBV cell line. These findings demonstrate that the CRISPR-Cas9 system is a potentially powerful tool capable of promoting a radical or “sterile” HBV cure.

Stronger enhancer II/core promoter activities of hepatitis B virus isolates of B2 subgenotype than those of C2 subgenotype.

Hepatitis B virus (HBV) genotype C causes prolonged chronic infection and increased risk for liver cancer than genotype B. Our previous work revealed lower replication capacity of wild-type genotype C2 than B2 isolates. HBV DNA replication is driven by pregenomic RNA, which is controlled by core promoter (CP) and further augmented by enhancer I (ENI) and enhancer II (ENII). DNA fragments covering these regulatory elements were amplified from B2 and C2 isolates to generate luciferase reporter constructs. As ENII is fully embedded in CP, we inserted HBV DNA fragments in the sense orientation to determine their combined activities, and in the antisense orientation to measure enhancer activities alone. Genotype B2 isolates displayed higher ENI+ENII+CP, ENII+CP, and ENII activities, but not ENI or ENI+ENII activity, than C2 isolates. The higher ENII+CP activity was partly attributable to 4 positions displaying genotype-specific variability. Exchanging CP region was sufficient to revert the replication phenotypes of several B2 and C2 clones tested. These results suggest that a weaker ENII and/or CP at least partly accounts for the lower replication capacities of wild-type C2 isolates, which could drive the subsequent acquisition of CP mutations. Such mutations increase genome replication and are implicated in liver cancer development.

Role of hepatitis B virus DNA integration in human hepatocarcinogenesis.

Liver cancer ranks sixth in cancer incidence, and is the third leading cause of cancer-related deaths worldwide. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, which arises from hepatocytes and accounts for approximately 70%-85% of cases. Hepatitis B virus (HBV) frequently causes liver inflammation, hepatic damage and subsequent cirrhosis. Integrated viral DNA is found in 85%-90% of HBV-related HCCs. Its presence in tumors from non-cirrhotic livers of children or young adults further supports the role of viral DNA integration in hepatocarcinogenesis. Integration of subgenomic HBV DNA fragments into different locations within the host DNA is a significant feature of chronic HBV infection. Integration has two potential consequences: (1) the host genome becomes altered ("cis" effect); and (2) the HBV genome becomes altered ("trans" effect). The cis effect includes insertional mutagenesis, which can potentially disrupt host gene function or alter host gene regulation. Tumor progression is frequently associated with rearrangement and partial gain or loss of both viral and host sequences. However, the role of integrated HBV DNA in hepatocarcinogenesis remains controversial. Modern technology has provided a new paradigm to further our understanding of disease mechanisms. This review summarizes the role of HBV DNA integration in human carcinogenesis.

Establishment of a stable cell line replicating hepatitis B virus DNA carrying the reverse transcriptase region derived from a clinical isolate

To establish a stable cell line that can replicate hepatitis B virus (HBV) DNA carrying the reverse transcriptase sequence derived from a clinical isolate. Nested PCR was used to amplify the HBV DNA fragment from the serum. The fragment was cloned into a plasmid that can support HBV replication in vitro by fragment substitution reaction (FSR), followed by the cloning of the neomycin expressing fragment downstream from HBV DNA. G418 selection was conducted after the transfection of HepG2 cells with the recombinant DNA. Real-time PCR and enzyme linked immunosorbent assay (ELISA) were used to screen stable cell lines that can replicate HBV DNA, and the replication of HBV DNA by the cell line was confirmed by using Southern blot analysis. Fragment nt55-1654 amplified from the serum DNA was substituted to the plasmid pLL, generating the plasmid p11. The neomycin fragment was cloned into p11, leading to the plasmid p11-neo, and p11-neo was confirmed to be HBV-replication-competent. A stable cell line named 3-10 that can replicate HBV DNA was obtained. A stable cell line was established that can replicate HBV DNA carrying the reverse transcriptase sequence derived from a clinical isolate. Real-time PCR plus ELISA may help to rapidly screen out stable cell lines replicating HBV DNA.

Putative roles of hepatitis B x antigen in the pathogenesis of chronic liver disease

Under most circumstances, hepatitis B virus (HBV) is noncytopathic. However, hepatocellular regeneration that accompanies each bout of hepatitis appears to be associated with increased integration of HBV DNA fragments expressing the virus encoded hepatitis B x antigen (HBxAg). Intrahepatic HBxAg staining correlates with the intensity and progression of chronic liver disease (CLD), and additional work has shown that HBxAg blocks immune mediated killing by Fas and by tumor necrosis factor alpha (TNFα). This is not only associated with the blockage of caspase activities by HBxAg, but also by the constitutive stimulation of hepatoprotective pathways, such as nuclear factor kappa B (NF-κB), phosphoinositol 3-kinase (PI3K), and beta-catenin (β-catenin). HBxAg also appears to promote fibrogenesis, by stimulating the production of fibronectin. HBxAg also stimulates the production and activity of transforming growth factor beta1 (TGFβ1) by several mechanisms, thereby promoting the profibrogenic and tumorigenic properties of this important cytokine. In addition, HBxAg appears to remodel the extracellular matrix (ECM) by altering the expression of several matrix metalloproteinases (MMPs), which may promote tumor metastasis. Hence, HBxAg appears to promote chronic infection by preventing immune mediated apoptosis of infected hepatocytes, by promoting the establishment and persistence of fibrosis and cirrhosis preceding the development of HCC, and by promoting the remodeling of EMC during tumor progression.

Replication of clinical hepatitis B virus isolate and its application for selecting antiviral agents for chronic hepatitis B patients

To establish a cell model harboring replicative clinical hepatitis B virus (HBV) isolates and evaluate its application in individualized selection of anti-HBV agents for chronic hepatitis B (CHB) patients. The full-length HBV genomic DNA from 8 CHB patients was amplified by polymerase chain reaction (PCR). All the patients were treated with lamivudine for at least seven months and finally became resistant to lamivudine. The amplified HBV DNA fragments were inserted into pHY106 vectors by Sap I digestion. The recombinant plasmids containing 1.1 copies of HBV genome were transiently transfected into Huh7 cell line, and the levels of HBsAg, HBeAg and intercellular HBV replicative intermediates were determined by ELISA and Southern blot analysis, respectively, with or without lamivudine and adefovir treatment. The antiviral treatment with adefovir was administered to the patients and analyzed in parallel. A total of 25 independent HBV isolates were obtained from the sera of 8 patients, each patient had at least two isolates. One isolate from each individual was selected and subcloned into pHY106 vector, including 5 isolates with YVDD mutation and 3 isolates with YIDD mutation. All recombinant plasmids harboring HBV isolates were transfected into Huh7 cells. The results indicated that HBV genome carried in HBV replicons of clinical HBV isolates could effectively replicate and express in Huh7 cells. Adefovir, but not lamivudine, inhibited HBV replication both in vitro and in vivo, and in vitro inhibition was dose-dependent. The novel method described herein enables individualized selection of anti-HBV agents in clinic and is useful in future studies of antiviral therapy for CHB.

Hepatitis B virus integration, fragile sites, and hepatocarcinogenesis

Chronic liver disease associated with long term hepatitis B virus (HBV) infection contributes importantly to the development of hepatocellular carcinoma (HCC). A salient feature of these chronic infections is the integration of subgenomic HBV DNA fragments into many different locations within the host DNA, suggesting that integration is random. Although this may promote genetic instability during liver regeneration which accompanies a bout of chronic liver disease, the actual role of integrated HBV DNA in hepatocarcinogenesis is uncertain. Importantly, most integration events retain the HBV open reading frame encoding the HBx antigen (HBxAg), which is the virus contribution to HCC. In addition, many integration events reported in the literature occur near or within fragile sites or other cancer associated regions of the human genome that are prone to instability in tumor development and progression. Genetic instability associated with integration potentially alters the expression of oncogenes, tumor suppressor genes, and microRNAs (miRNAs) that may contribute importantly to tumorigenesis. If so, then selected integration events may alter pathways that are rate limiting in hepatocarcinogenesis, thereby providing targets with diagnostic/prognostic potential and for therapeutic intervention.

Simultaneous Detection of Precore/Basal Core Promoter Mutations in Hepatitis B Virus Using Arrayed Primer Extension

Hepatitis B is a major disease that causes serious public health problems worldwide. The loss of HBeAg expression due to point mutations or single nucleotide polymorphisms (SNPs) in the precore/basal core promoter region of the hepatitis B virus (HBV) is associated with hepatocellular cirrhosis and carcinoma. Simultaneous screening for these mutations is strongly advocated for monitoring disease development in HBV-infected patients. The aim of this study is to apply arrayed primer extension (APEX) for the detection of HBV SNPs at the precore/basal core promoter. We optimized APEX for simultaneous detection of eight potential sites of SNPs in the precore/basal core promoter region of HBV. The precore/basal core promoter regions of HBV from 36 HBV-infected patients were amplified by PCR. After purification and DNA fragmentation, the short, single-stranded HBV DNA fragments were allowed to hybridize with the oligonucleotides corresponding to the sites of SNPs immobilized on glass slides, followed by incorporation of different fluorescently labeled dideoxynucleotides. This allows fast and unequivocal discrimination between wild-type and mutant genotypes with high dideoxy-nucleotide incorporation efficiency, sensitivity, and specificity. The coexistence of both genotypes was also detected; this was undetected by DNA sequencing. The simultaneous detection of SNPs in HBV precore/basal core promoter by APEX enables large-scale diagnostic analysis, which can be extended to the whole HBV genome.

Hybridization biosensor using di(2,2 ′-bipyridine)osmium (III) as electrochemical indicator for detection of polymerase chain reaction product of hepatitis B virus DNA

A novel hepatitis B virus (HBV) DNA biosensor was developed by immobilizing covalently single-stranded HBV DNA fragments to a gold electrode surface via carboxylate ester to link the 3 ′-hydroxy end of the DNA with the carboxyl of the thioglycolic acid (TGA) monolayer. A short-stranded HBV DNA fragment (181 bp) of known sequence was obtained and amplified by PCR. The surface hybridization of the immobilized single-stranded HBV DNA fragment with its complementary DNA fragment was evidenced by electrochemical methods using [Os(bpy) 2Cl 2] + as a novel electroactive indicator. The formation of double-stranded HBV DNA on the gold electrode resulted in a great increase in the peak currents of [Os(bpy) 2Cl 2] + in comparison with those obtained at a bare or single-stranded HBV DNA-modified electrode. The mismatching experiment indicated that the surface hybridization was specific. The difference between the responses of [Os(bpy) 2Cl 2] + at single-stranded and double-stranded DNA/TGA gold electrodes suggested that the label-free hybridization biosensor could be conveniently used to monitor DNA hybridization with a high sensitivity. X-ray photoelectron spectrometry technique has been employed to characterize the immobilization of single-stranded HBV DNA on a gold surface.

HBV integrants of hepatocellular carcinoma cell lines contain an active enhancer.

Hepatitis B virus (HBV) infection is a major risk factor worldwide for the development of hepatocellular carcinoma (HCC). Integrated HBV DNA fragments, often highly rearranged, are frequently detected in HCC. In woodchuck, the viral enhancer plays a central role in hepatocarcinogenesis, but in humans the mechanism of HBV oncogenesis has not been established. In this study we investigated the status of the viral enhancer in two human HCC cell lines, Hep3B and PLC/PRF/5 each containing one or more integrated HBV DNA fragments. Active enhancer was defined by virtue of its protein occupancy as determined by genomic in vivo DMS footprinting. In PLC/PRF/5 cells, the HBV DNA was integrated in a cellular gene at chromosome 11q13, at a locus reported to be amplified in many tumors. We show here that in both cell lines, the integrated HBV DNA fragments contain an active enhancer-I. In particular, the occupation of the two previously defined basic enhancer elements, E and EP, was prominent. While in both cell lines the same protein binds to the EP elements, the E element, however, is occupied in a cell-line specific manner. In PLC/PRF/5 but not Hep3B, the prominent binding of an undefined protein was detected. Our data suggest that this protein is likely to be the fetoprotein transcription factor (FTF). The finding that enhancer sequences are conserved and functional in different cell lines suggests a selection pressure for their long-term maintenance. We therefore propose that the HBV enhancer-I might play a role in hepatocellular carcinogenesis.

Using Silane Coupling Reagents in Fabrication of DNA Arrays on Glass Support

Two silane reagents were used to modify the surface of glass slide. The modified surfaces were characterized using X-ray photoelectron spectroscopy (XPS). DNA arrays generated by polymerase chain reaction (PCR) from the genomic DNA of hepatitis B virus (HBV) were spotted on the slides by capillary tube. Such slides were applied to detect the HBV DNA fragment by hybridization with fluorescence-labeled probe.

A novel negative cis-regulatory element on the hepatitis B virus S-(+)-strand.

Hepatitis B virus (HBV) has a double-stranded DNA genome. The minus-strand contains coding regions for all known HBV proteins and most of the cis-regulatory elements. Little is known about transcription from the S-(+)-strand and its regulation. Thus, the presence of regulatory elements located on the S-(+)-strand was investigated by inserting nt 1038-1783 of HBV in both orientations between the human cytomegalovirus (HCMV) promoter and a luciferase gene. Transfection experiments revealed that the plasmid containing this HBV DNA fragment in an orientation allowing expression from the S-(+)-strand (antisense) led to inhibition of luciferase gene expression compared to the plasmid containing this sequence in an orientation that allows gene expression from the L-(-)-strand (sense). Deletion analyses delimit the sequence essential for the inhibitory effect to a 150 bp region that also carries part of the enhancerII/core promoter complex. However, the possible influence of this regulatory element has been excluded in various experiments. The repressing HBV sequence acts in an orientation- and position-dependent manner; no inhibition was observed when this DNA element was inserted upstream of the HCMV promoter or downstream of the luciferase gene. Northern blot analyses revealed reduced luciferase mRNA steady-state levels in cells transfected with constructs containing the essential HBV sequence in antisense orientation compared to plasmids containing this sequence in sense orientation. Since nuclear run-on experiments showed similar transcription initiation rates with these plasmids, the diminished luciferase mRNA steady-state levels must be due to altered stabilities, suggesting that nt 1783-1638 of HBV encode an RNA-destabilizing element.

Clinical implications of mutations C-to-T1653 and T-to-C/A/G1753 of hepatitis B virus genotype C genome in chronic liver disease.

Among many mutational "hot spots" on hepatitis B virus (HBV) genome, A-to-T1762 and G-to-A1764 within the core promoter have been underscored in view of disease association as well as viral expression/replication. Although to a lesser extent, C-to-T1653 and T-to-V(C/A/G)1753 were also noteworthy in our previous study. To assess the clinical significance of these mutations, we determined the nucleotide sequence of an HBV DNA fragment covering these sites in HBsAg-positive blood donors (n = 160) and patients with chronic hepatitis (n = 66), liver cirrhosis (n = 45), and hepatocellular carcinoma (n = 58), most of whom were infected with genotype C HBV (subtype adr). In cases where HBe antigen was positive, the frequency of T1653 and/or V1753 showed a striking increment from chronic hepatitis patients (18%) to liver cirrhosis and/or hepatoma patients (82%), whereas that of T1762/A1764 was already high in chronic hepatitis patients (76%). In HBe antigen-negative cases, by contrast, significant difference in the frequency of T1653/V1753 mutants was found between blood donors (22%) and chronic hepatitis patients (67%). Our results suggest that T1653/V(particularly C)1753 mutants are more closely associated than T1762/A1764 with the progression of liver disease from chronic hepatitis to cirrhosis in HBe antigen-positive patients. A system of site-directed mutagenesis PCR RFLP was constructed to diagnose T1653 and C/A1753 more conveniently. Detecting T1653 and C/A1753 by this method would contribute to the differential diagnosis of HBV-associated liver disease.

Sensitive method for identification of minor hepatitis B mutant viruses

There is increasing evidence for selection of hepatitis B virus (HBV) mutants during the natural course of infection, after immunization, and during interferon therapy. These mutants can prolong viral persistence, escape immune-mediated elimination, and influence the clinical sequelae of infection. Here we describe a sensitive and reliable method that allows detection of HBV mutants within a heterogeneous virus population. The method consists of amplification of HBV DNA with vector-HBV hybrid primers, efficient and enzyme-free cloning of the amplified DNA fragment, identification by hybridization with synthetic oligonucleotide probes, and sequencing of the cloned DNA fragments. We demonstrate that up to 10 5 HBV DNA fragments can be obtained in cloned form from a single amplification reaction, the ratio of HBV pre-C “wild-type” and mutant sequences of heterogeneous populations can be determined, and very minor virus subpopulations can be identified. The method can be applied to other viral or nonviral nucleic acid sequence variation studies and should help to resolve the role of mutant and wild-type viruses in pathogenesis and the forces driving their selection.