Infection with the hepatitis B virus (HBV) leads to

Transcription

1 Intrahepatic Levels and Replicative Activity of Covalently Closed Circular Hepatitis B Virus DNA in Chronically Infected Patients Andreas Laras, 1 John Koskinas, 1 Evangelini Dimou, 1,2 Ageliki Kostamena, 1 and Stephanos J. Hadziyannis 1,2 Hepatitis B virus (HBV) covalently closed circular DNA (cccdna) is responsible for viral persistence in the natural course of chronic HBV infection and during prolonged antiviral therapy and serves as the template for the production of HBV pregenomic RNA (pgrna), the primary step in HBV replication. In this study, we have developed and applied sensitive and specific quantitative real-time polymerase chain reaction (PCR) assays for the measurement of intrahepatic concentration, pgrna production, and replicative activity of cccdna in liver biopsy samples from 34 non-treated patients with chronic hepatitis B (CHB); 12 hepatitis B e antigen (HBeAg)( ) and 22 HBeAg( ). Median copy number for cccdna was 1.5 per cell and for pgrna significantly higher, 6.5 copies per cell, with a good correlation between cccdna and pgrna levels in all samples. In HBeAg( ) patients, median values of cccdna and pgrna levels were 10-fold and 200-fold lower than in HBeAg( ), respectively, reflecting the differences in viral activity and clinical characteristics of the two groups. Furthermore, the replicative activity of intrahepatic cccdna was significantly lower in HBeAg( ) patients harboring mutant HBV strains than in HBeAg( ) patients: median 3.5 versus 101 pgrna copies per cccdna molecule. In conclusion, the levels of both HBV cccdna, a marker of HBV persistence, and pgrna, an indicator of viral replication, in the liver of chronically infected patients correlate with viral activity and the phase of HBV infection. The combined measurement of cccdna and pgrna levels provides valuable information on the presence and replicative activity of intrahepatic HBV cccdna. (HEPATOLOGY 2006;44: ) Infection with the hepatitis B virus (HBV) leads to either acute, self-limiting, or fulminant hepatic disease, or to chronic hepatitis B (CHB) infection with a wide spectrum of viral activity, clinical manifestations, and disease progression. 1-3 The factors and molecular events that influence the course and the outcome of chronic infection remain poorly understood. Persistent Abbreviations: HBV, hepatitis B virus; CHB, chronic hepatitis B; cccdna, covalently closed circular DNA; pgrna, pregenomic RNA; CP, core promoter; HBeAg, hepatitis B e antigen; BCP, basic core promoter; rcdna, relaxed circular DNA; RT-PCR, reverse transcription polymerase chain reaction. From the 1 Hepatitis Research Laboratory, Athens University School of Medicine; and 2 Henry Dunant Hospital, Athens, Greece. Received November 17, 2005; accepted May 31, Gilead Sciences has provided financial support to the Hepatitis Research Laboratory. Address reprint requests to: Professor Stephanos J. Hadziyannis, Department of Medicine and Hepatology Unit, Henry Dunant Hospital, Mesogion 107, Athens Greece. hadziyannis@ath.forthnet.gr; fax: (30) Copyright 2006 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience ( DOI /hep Potential conflict of interest: Nothing to report. HBV infection of the hepatocyte is characterized by the presence of a covalently closed circular (ccc) DNA episome, whereas the degree of viral replicative activity is primarily determined by the transcriptional state of cccdna molecules and the production of pregenomic (pg) RNA. 4-6 On entry of the HBV virion into the hepatocyte, the viral genome is translocated to the nucleus and converted into a supercoiled cccdna viral minichromosome. 7-9 The cccdna episome is the template for the HBV mrna transcripts; and the 3.5-kb pg RNA, template for reverse transcription and synthesis of the viral genome. Transcription of pgrna from cccdna is a key event in HBV replication, and the process is under the control of the HBV core promoter (CP), 10 which directs the synthesis of two species of overlapping 3.5-kb viral transcripts that only differ at their 5 initiation sites. 11,12 Translation of the longer precore (prec) mrna leads to the synthesis of the precore protein, precursor of the HBV e antigen (HBeAg). 13 The shorter pg RNA serves as mrna for the core and the polymerase genes and, after encapsidation, as 694

2 HEPATOLOGY, Vol. 44, No. 3, 2006 LARAS ET AL. 695 the template for reverse transcription to generate viral DNA. Intracellular cycling of DNA-containing nucleocapsids to the nucleus is necessary for cccdna pool maintenance. 9,14 During the course of chronic HBV infection and during prolonged antiviral therapy, development and selection of mutations in the HBV genome may become clinically relevant and influence the outcome of infection CP mutations clustering in the basic CP (BCP) region are commonly found among chronically infected patients. 18,19 CP mutations may affect HBV gene expression, replication, and pathogenicity by altering the levels and balance between pgrna and precore mrna production and affecting the transcriptional activity of HBV cccdna molecules. HBV cccdna is believed to be responsible for viral persistence and reactivation However, the factors that determine the transcriptional state of cccdna in hepatocytes are not well understood, and widespread HBV mutant strains have different transcriptional and replicative activities. 20,23,27,28 Therefore, measuring in vivo pgrna levels in conjunction with cccdna levels could provide a more precise method for evaluating HBV persistence and activity in infected patients than measuring cccdna alone. Despite the generation and publication of methodology on HBV cccdna detection, few in vivo data are currently available from human liver samples, and most of the existing knowledge is derived from animal models Even less in vivo information is available on cccdna replicative activity and pgrna production, 22,37 because most HBV expression studies have been performed in vitro or on heterologous systems. We have designed and used quantitative assays for the measurement of both concentration and, for the first time, the replicative activity of intrahepatic cccdna in liver biopsy samples of patients with CHB. Our data show that the combined measurement of cccdna and pgrna levels provides valuable information by monitoring the presence, as well as the activity of intrahepatic HBV cccdna in chronically infected patients. Patients and Methods Patients and Biological Samples. We studied 34 patients with CHB; 12 HBeAg( ), 7 men and 5 women, median age 36 (range, 18-58) years and 22 HBeAg( ), 12 men and 10 women, median age 42 (27-65) years. All patients had active liver disease with persistent biochemical activity [serum alanine aminotransferase (ALT) levels 2 the upper limit of normal] and had been on longterm follow-up on an outpatient basis. Degree of hepatic inflammation and fibrosis was assessed according to Ishak et al. 40 : 7 (21%) had established cirrhosis [1 HBeAg( ), 6 HBeAg( )], 17 (50%) had fibrosis score 3 [4 HBeAg( ), 13 HBeAg ( )], and the mean necro-inflammatory score was 8 (range, 4-14). All patients were negative for serological markers for hepatitis D virus, hepatitis C virus, and HIV infection. None was on treatment or had received nucleoside analog treatment in the past. None was consuming alcohol or taking immunosuppressive drugs. Serological markers of HBV infection, hepatitis B surface antigen, HBeAg, and anti-hbe were tested by radioimmunoassay (Sorin Biomedica S.p.A, Saluccia, Italy). Liver biopsies and synchronous sera samples were stored at 70 C until analysis. This study was approved by the Ethics Committees of the Evgenidion Hospital of Athens University and Henry Dunant Hospital. HBV cccdna Detection and Quantitation. Liver biopsy material from each patient was homogenized and divided into two parts, one for DNA and one for RNA/ DNA extraction. Total liver DNA was extracted from biopsy samples using the Qiamp DNA Mini Kit (QIA- GEN GmbH, Germany). Real-time polymerase chain reaction (PCR) was performed with the LightCycler FastStart DNA Master Hybridization Probes kit (Roche Diagnostics GmbH, Mannheim, Germany), using 3 antisense primer BC1 (5 -GGAAAGAAGTCAGAAG- GCAA, nt ) and 5 primers CCC (5 - GTGCCTTCTCATCTGCCGG nt ) or PGP (5 -CACCTCTGCCTAATCATC nt ) that specifically detect cccdna and total HBV DNA, respectively (Fig. 1). FRET hybridization probes 0.4 mmol/l hbvlc (5 -TGGAGGCTTGAACAGTAGGACATGA- AC nt ) labeled with LC Red 640 at the 5 terminus and 0.2 mmol/l hbvfl (5 -CYAAAGCCAC- CCAAGGCACAGC nt ) labeled with fluoresceine at the 3 terminus (Fig. 1). After 10 minutes incubation at 95 C amplification was performed for 45 cycles (95 C 10 seconds, 52 C 16 seconds, and 72 C 10 seconds). Plasmid-Safe DNase (Epicentre, Madison, WI) was used according to the manufactures instructions, in testing designed to validate the specificity of the assay. Cell number in liver biopsies was estimated by measuring -globin gene levels using the LightCycler Control Kit DNA (Roche Diagnostics GmbH, Mannheim, Germany); results were normalized to cells. Pregenomic RNA Quantitative Detection/Transcript-Specific RT-PCR. Total liver RNA (and subsequently total liver DNA) was extracted from liver biopsy samples using a modified guanidinium thiocyanate-phenol procedure (TriPure, Roche Diagnostics, GmbH). RNA samples were treated with RQ1 RNase-free DNase (Promega, Madison, WI, USA) at 1 U/ g nucleic acid for

3 696 LARAS ET AL. HEPATOLOGY, September 2006 Fig. 1. Schematic representation of the hepatitis B virus (HBV) double-stranded relaxed circular (rc) DNA and covalently closed circular (ccc) DNA and organization of the core promoter and precore/core regions. Direct repeats (DR) 1 and 2 used in HBV replication are shown as black boxes. Regulatory elements that constitute the core promoter and their corresponding nucleotide positions are shown, negative regulatory element (NRE) nt , core upstream regulatory sequences (CURS) nts and basic core promoter (BCP) nts Precore mrna and pregenomic RNA transcripts are shown with horizontal arrows indicating the origin and direction of transcription. Real-time polymerase chain reaction (PCR) primers used for cccdna detection (CCC and BC1) and for the transcript-specific reverse transcription (RT)-PCR assay (M3, PCP, PGP, and BC1) are shown as horizontal arrows. The position of the LightCycler hybridization probes hbvlc and hbvfl is also shown. Shaded triangles indicate the translation initiation codons for precore and core and the open triangle the polyadenylation (PolyA) signal for HBV RNA transcripts. 1 hour at 37 C, to remove contaminating DNA. One microgram liver RNA was used for cdna synthesis with antisense primer BC1 and M-MLV reverse transcriptase (RT). Extracted liver RNA (1 g) was incubated for 40 minutes at 42 C in 60 L reaction mixture containing 50 pmol cdna primer, 50 mmol/l Tris (ph 8.3), 75 mmol/l KCl, 3 mmol/l MgCl 2, 10 mmol/l dithiothreitol, 1 mmol/l dntps, 20 U Rnasin (Promega), and 300 U M-MLV RT (Promega). The cdna product was used in each of three separate amplification reactions with BC1 as the common 3 primer and 5 primers: (1) PCP (5 - GGTCTGCGCACCAGCACC nt ) for the specific detection of precore mrna transcripts, (2) PGP (5 -CACCTCTGCCTAATCATC nt ) for monitoring total CP-directed transcription, and (3) M3 (5 -CTGGGAGGAGTTGGGGGAGGAGATT nt ) for detecting contaminating HBV DNA molecules (Fig. 1). FRET hybridization probes and realtime PCR conditions were as described. HBV cccdna replicative activity was expressed as molecules of pgrna synthesized per HBV cccdna molecule. DNA extracted subsequently was used to estimate cell number as described. Serum HBV DNA Extraction, PCR Amplification, and Sequencing. HBV DNA was extracted from 200 L serum amplified and sequenced as described previously. 22 Serum cccdna and total HBV DNA were quantified by real-time PCR as described. Statistical Analysis. Statistical analyses were performed using SPSS10.0 (Chicago, IL). Correlation between two variables was tested by using Pearson s analysis after logarithmic transformation. Statistical significance was denoted as P.05. Results Specificity and Sensitivity of cccdna and pgrna Quantitative Assays. A major concern in designing and evaluating cccdna-specific detection assays is the complex structure of the HBV genome, which may result in nonspecific background signal compromising the integrity of data. Because the complexity and heterogeneity of HBV genomic DNA is not reflected in cloned HBV sequences, we have tested the performance of the assay on HBV DNA extracted from serum and liver biopsy samples. Liver biopsy specimens and synchronous serum samples randomly selected from three HBeAg( ) (patients 2, 5, and 6) and six HBeAg( ) (patients 13, 15, 20, 25, 27, and 34) patients were used initially to determine the specificity of the cccdna detection assay. Total HBV DNA and cccdna levels were measured in liver and serum samples, and the results are shown in Table 1. Serum HBV DNA ranged from to copies per milliliter; cccdna was detected in seven of nine serum samples tested and ranged from to copies per milliliter. Corresponding intrahepatic total HBV DNA levels ranged from to per cells and cccdna, detected in all liver samples ranged from to per cells.

4 HEPATOLOGY, Vol. 44, No. 3, 2006 LARAS ET AL. 697 Table 1. Specific Detection of HBV Total DNA and cccdna in Synchronous Serum and Biopsy Samples From Nine Randomly Selected Patients Patient Number HBeAg Status Serum HBV DNA (copies/ml) Liver HBV DNA (copies/10 6 cells) Total DNA cccdna % cccdna Total DNA cccdna % cccdna UN UN Abbreviation: UN, undetectable. Total serum HBV DNA levels were 1,000-fold (800- to 6,500-fold) higher than the corresponding serum cccdna levels, which represents 0.1% (range, 0.02%- 0.12%) of serum HBV DNA. In contrast, high levels of cccdna were detected in all liver samples, representing a significant proportion of total intrahepatic HBV DNA, 6% on the average (range, 0.65% to 22%), with total HBV DNA levels being 17-fold (5-fold to 154-fold) higher. Although other investigators have reported the presence of HBV cccdna 33 and naked HBV nucleic acids 41,42 in the bloodstream, it could still be argued that measured serum cccdna is a result of nonspecific detection of the assay. Even in this case, a background of nonspecific cccdna detection of 1:1,000 molecules would not affect the integrity of the liver data. To dismiss this possibility, the specificity of the cccdna detection assay was further validated by treatment with Plasmid-Safe DNase, which selectively degrades linear DNA or circular single-stranded DNA without affecting closed or nicked circular doublestranded DNA. Total HBV DNA samples extracted from four liver biopsies and synchronous serum samples were subjected to DNase treatment, and detected cccdna levels were compared with those measured in mock-treated samples (Table 2). HBV cccdna levels were resistant to Patient Number Table 2. Effect of Plasmid-Safe DNase Treatment on cccdna-specific Detection in Synchronous Biopsy and Serum Samples HBeAg Status Liver HBV cccdna (copies/10 6 cells) DNase Treated Mock Treated Serum HBV cccdna (copies/ml) DNase Treated Mock Treated DNase treatment in all samples tested, demonstrating the high specificity of our cccdna detection assay. The sensitivity of the real-time assay was determined using serially diluted samples of serum and liver HBV DNA. The lower limit of detection was 10 copies/assay for total HBV DNA detection and 100 copies/assay for cccdna detection, with a dynamic range of 6 orders of magnitude. This is in part attributable to a less efficient amplification by the cccdna-specific primers, which amplify a longer DNA fragment than the primers used in the detection of total HBV DNA (420 bp vs. 149 bp). Taking into account the cell number of the liver biopsy specimens and necessary sample dilutions, intrahepatic detection limits were and copies/cell, respectively, for total HBV DNA and cccdna assays. The specificity of the methodology used in the transcript-specific RT-PCR assay for the differential detection of precore mrna and pgrna transcripts has been previously described. 22 Application of RT-PCR technology has improved the sensitivity of the method to 10 copies/ assay for either precore mrna or total BCP-directed transcription. Quantitative Detection of cccdna in the Liver. HBV cccdna levels were measured in all liver biopsy specimens from 22 HBeAg( ) and 12 HBeAg( ) patients. Total liver DNA was analyzed by template-specific real-time PCR for the presence of cccdna and total intracellular HBV DNA, which also includes immature and mature viral genomic DNA. Cell number in the liver biopsy samples was calculated by measuring -globin gene levels, and results were normalized to cells (number of copies/cell is also given to facilitate comparisons with other studies). The median copy number of cccdna per cells was ranging from to (median, 1.46; range, copies/cell). High levels of cccdna were detected in HBeAg( ) patients with a

5 698 LARAS ET AL. HEPATOLOGY, September and ranging from to (median, 266; range, 96-8,730 copies/cell). In contrast, low levels of pgrna were detected in the liver of HBeAg( ) patients, with a median of , ranging from to (median, 1.4; range, copies/cell) (Fig. 2). Precore mrna transcripts were detected in only 22 patients, and their levels were significantly lower than the corresponding pgrna levels. Precore mrna was measured in all HBeAg( ) patients with a median value of and ranging from to (median, 2.4; range, copies/cell), representing Fig. 2. Intrahepatic cccdna, pgrna and total intracellular HBV DNA levels in HBeAg( ) (open squares) and HBeAg( ) (closed circles) patients. median of , ranging from to (median, 3.0; range, copies/cell). Significantly lower levels of cccdna were detected in HBeAg( ) patients with a median of , ranging from to (median, 0.31; range, copies/cell) (Fig. 2). The levels of total intracellular HBV DNA in these patients had a median copy number of , ranging from to per cells (median, 31; range, ,890 copies/cell). Total intracellular HBV DNA levels were higher in HBeAg( ) patients, with a median of (range, ) than in HBeAg( ) patients, with a median of (range, ) per cells (Fig. 2). A strong correlation was seen between intrahepatic total HBV DNA and cccdna levels (r 0.945) (Fig. 3A); however, the percentage of intrahepatic HBV DNA in the form of cccdna is inversely related to the total intrahepatic and serum HBV DNA levels (partially shown in Table 1). Quantitative Analysis of pgrna Transcripts in the Liver. Total liver RNA was analyzed by transcript-specific real-time RT-PCR, which allows specific detection and quantification of precore mrna and simultaneously monitors total RNA transcription (precore mrna plus pgrna) directed by the core promoter in the liver of infected patients. The levels of pgrna transcripts were calculated by subtracting precore mrna levels from total CP-directed transcription. HBV pgrna transcript levels per cells had a median copy number of ranging from to (median, 6.5; range, ,730 copies per/cell). High levels of pgrna transcripts were measured in HBeAg( ) patients having a median of 2.7 Fig. 3. Correlation between various parameters measured in liver biopsy samples of hepatitis B e antigen (HBeAg)( ) (open squares) and HBeAg( ) patients (closed circles). (A) Intrahepatic total hepatitis B virus (HBV) DNA versus cccdna, correlation coefficients: r for HBeAg( ) and r for HBeAg( ) patients. (B) Pregenomic RNA vs cccdna, correlation coefficients: r for HBeAg( ) and r for HBeAg( ) patients. (C) Pregenomic RNA versus intrahepatic total HBV DNA, correlation coefficients: r for HBeAg( ) and r for HBeAg( ) patients.

6 HEPATOLOGY, Vol. 44, No. 3, 2006 LARAS ET AL. 699 approximately 1% of total CP-directed transcription. In contrast, precore mrna was detectable in only 10 of 22 HBeAg( ) patients, with a median of and ranging from to (median, 0.18; range, copies/cell). Eleven of 12 HBeAg( ) patients harboring the A1762T/G1764A mutation had undetectable precore mrna levels. Four patients with either one of A1762T, G1764A, or other mutations in the BCP promoter had intermediate precore mrna levels, whereas five HBeAg( ) patients without any BCP mutations expressed high precore mrna levels. The levels of pgrna in the liver were found to correlate very well with cccdna (r 0.863) (Fig. 3B) and the levels of total intrahepatic HBV DNA (r 0.964) (Fig. 3C). Generally good correlation was seen between all HBV nucleic acid replication parameters. Serum HBV DNA levels show significant correlation with intrahepatic cccdna levels (r 0.744) and highly correlate with intrahepatic pgrna and total HBV DNA levels (r and r 0.840, respectively). Replicative Activity of Intrahepatic cccdna. Production of pgrna from transcriptionally active cccdna molecules is a key step in HBV replication and therefore a measure of the replicative activity of intrahepatic cccdna. The simultaneous measurement of cccdna and pgrna allows us to measure for the first time the activity of cccdna molecules in the liver of patients with CHB. Good correlation between cccdna and pgrna levels was found in all samples tested (Fig. 3B); however, considerable differences were found in the median values for cccdna and pgrna levels between the two groups (Fig. 2). In HBeAg( ) patients median cccdna levels were 10-fold ( vs ) and median pgrna levels 200-fold ( vs ) lower than in HBeAg( ) patients. Thus the replicative activity of the cccdna intermediate, calculated as the number of pgrna copies produced per cccdna molecule, was significantly different between the HBeAg( ) and the HBeAg( ) patients. Median replicative activity of cccdna in HBeAg( ) patients was 101 (range, ) versus 3.5 (range, ) in HBeAg( ) patients. Most (12 of 22) HBeAg( ) patients had very low replicative activity, less than 5 pgrna transcripts per cccdna molecule, whereas only 6 of 22 patients had replicative activity greater than 15 pgrna/cccdna (Fig. 4). The reduced CP transcriptional activity of cccdna in HBeAg( ) patients can be at least in part attributed to the accumulation of mutations in the CP and precore regions of the viral genome. Although all HBeAg( ) patients had wild-type CP and precore sequences, HBeAg( ) patients were found to harbor the precore stop codon mutation Fig. 4. HBV cccdna Replicative Activity (pgrna transcripts produced per cccdna molecule) in HBeAg( ) (open squares) and HBeAg( ) (closed circles) patients. G1896A and additional mutations in the CP region, most prevalent being the double A1762T/G1764A BCP mutation. Discussion Persistent hepatocyte infection by HBV is achieved by the establishment and maintenance of a stable cccdna pool in the nuclei of infected cells. 7-9 Replenishment of the cccdna nuclear reservoir, as well as initiation of viral replication, require the synthesis of pgrna transcripts from episomal viral genomes. 4-6,14 In this study, we have developed and applied sensitive assays for the quantitative analysis of HBV cccdna and pregenomic RNA in liver biopsy samples of chronically HBV infected patients, which enable us to measure both the concentration and the replicative activity of intrahepatic cccdna. In designing the cccdna-specific real-time PCR detection assay, we have positioned the amplification primers at conserved regions flanking the direct repeats DR1 and DR2 and the nicks in both ( ) and ( ) strands (Fig. 1). However, that both 3 primer and FRET detection probes are placed downstream from DR1 and that probes have ( ) polarity are critical, to avoid a nonspecific signal resulting from asymmetric amplification of either the ( ) or ( ) strands, which would obscure the measurement of the low-concentration intrahepatic cccdna component. Perhaps for this reason other studies report significant background from relaxed circular (rc) DNA in their experiments, necessitating enzymatic DNase digestion before cccdna detection in their methodology. 32,34 Our methodology is specific by design, and, as we have shown, it does not depend on DNase removal of other HBV DNA molecules (Table 2).

7 700 LARAS ET AL. HEPATOLOGY, September 2006 Transcript-specific detection of pgrna and precore mrna is achieved, first, by positioning the 3 primer downstream from the common polyadenylation site, unique sequences present at the 5 end of the CP produced 3.5-kb RNAs allow distinction between them and the rest of the HBV transcripts (Fig. 1). Second, specific detection of the longer precore transcript is accomplished by positioning the 5 primer in the small region between the precore mrna and the pgrna start sites. A second 5 primer positioned downstream from both start sites monitors total CP-directed transcription (Fig. 1). Our in vivo study demonstrated the presence of HBV cccdna in all included patients with CHB at low median levels of approximately 1 copy/cell, but exhibiting a wide dynamic range of four orders of magnitude. In general, cccdna levels correlate with HBeAg status and viral activity. HBeAg( ) patients with CHB had high cccdna copy numbers, approximately 1 to 40 copies/cell, and high levels of total intracellular HBV DNA (95-9,890 copies/cell) and serum HBV DNA ( copies/ml). A far more complex situation exists within the HBeAg( ) group, where median cccdna levels are 10- fold lower compared with HBeAg( ) patients. Intrahepatic cccdna levels show significant variability ( copies/cell), consistent nonetheless with the heterogeneity of this group in terms of viral genomic sequences, replication, and disease activity. In HBeAg( ) patients with low viremia, chronic infection appears to be maintained by a very low cccdna copy number ( 0.1 copies per cell), in agreement with the study of Zhang et al. 38 The accumulation of mutations in the precore region and the BCP appears to adversely affect intrahepatic cccdna levels in HBeAg( ) patients, suggesting that the cccdna pool might be affected by pgrna production. This is further supported by the good correlation of intracellular cccdna and pgrna levels (r 0.863). This could be a result of specific processes regulating HBV replication and intracellular trafficking of nucleocapsids 43,44 or it simply could be attributable to lower pgrna levels, which would result in a reduced pool of precursor genomic molecules available for the replenishment of nuclear cccdna. The levels and range of intrahepatic cccdna, as well as total liver HBV DNA, in HBeAg( ) and HBeAg( ) patients in this study are consistent with and support the available data in the literature. 33,34 However, serum cccdna levels were found to be lower than those reported by Wong et al. 33 (0.1% vs. 3.5%), perhaps reflecting differences in methodology or the characteristics of patients and viral strains in the two studies. Although pregenomic RNA is important both for viral production and replenishment of the cccdna reservoir, 9,14 little in vivo information is currently available. 22 This is the first report that accurately measures in vivo pgrna levels in the liver of patients with CHB. Our study showed an extremely wide range of pgrna levels (0.01-8,700 copies/cell), which nonetheless correlate with viral activity and phase of HBV infection, as is the case with cccdna levels. Pregenomic RNA levels were consistently high at the early HBeAg( ) phase and more variable but significantly lower (200-fold) at the later HBeAg( ) phase. PgRNA levels were significantly higher than the corresponding levels of cccdna, 90-fold higher in HBeAg( ) patients and fivefold higher in HBeAg( ) patients. Therefore, measurement of pg RNA levels in biopsy samples may provide more accurate information on viral replication as well as higher sensitivity, particularly in HBeAg( ) patients with low viremia and cccdna levels. Furthermore, because precore mrna contribution to CP-directed transcription is low, it may be possible to perform a single assay for monitoring pgrna levels; however, a larger number of samples should be analyzed before making this recommendation. The observed differences and variability of pgrna levels can be attributed only in part to differences in the cccdna template content in the liver of chronic HBV patients, and they must also be accounted for by changes in the transcriptional activity of intrahepatic cccdna molecules. A unique result of this study is the assessment of the replicative activity of intrahepatic cccdna by simultaneously measuring cccdna and its pgrna transcript levels in the same tissue sample of patients with CHB. This allows us to evaluate the transcriptional and replicative activity of cccdna molecules in vivo. HBV cccdna replicative activity was high in all HBeAg( ) patients harboring wild-type HBV strains. The intensive pgrna production coupled with high cccdna levels in this group results in a highly productive infections with median viremia copies/ml serum. In contrast, in HBeAg( ) patients, cccdna replicative activity was dramatically lower. Furthermore, although the correlation between cccdna, pgrna, and viremia is significant, the correlation coefficients are relatively low, suggesting that other factors, such as the accumulation of mutations and changes in the host immune response, may significantly affect HBV production and disease activity in the HBeAg( ) phase. Mutations in the BCP have been shown to suppress precore mrna production and alter the ratio between pgrna and precore mrna synthesis In this study, BCP mutants had low or undetectable precore mrna levels and lower replicative activity than wild-type strains, as did HBV G1896A precore mutants. Given the variabil-

8 HEPATOLOGY, Vol. 44, No. 3, 2006 LARAS ET AL. 701 ity in viral activity characterizing HBeAg( ) CHB, significant fluctuations in the cccdna pool or its replicative activity may occur during the course of infection. However, to what extent activation or sequestering of cccdna activity is determined by mutations accumulating in the CP promoter or other regions of the HBV genome remains undetermined. High levels of viral production may not necessitate a relative increase in the cccdna pool but rather enhanced levels of transcriptional activity mediated by other modulatory factors. In agreement with this hypothesis is the observation that three HBeAg( ) patients had relatively high replicative activity ( 20) in the absence of obvious differences in cccdna levels and mutation profile. Reduced replicative activity and inefficient replenishment of the nuclear cccdna reservoir could explain differences in the intrahepatic cccdna levels between HBeAg( ) and HBeAg( ) patients reported in this and other studies. 33,34 Collectively our data demonstrate that differences in the natural course of HBV infection between HBeAg( ) and HBeAg( ) patients are not limited to decreased intrahepatic cccdna levels. On the contrary, although a significant reduction occurs in cccdna concentration, a much stronger decrease is seen in corresponding intrahepatic pgrna levels, reflecting a markedly reduced replicative activity of cccdna in HBeAg( ) patients. In conclusion, in the liver of chronically infected patients, the levels of both HBV cccdna, a marker of HBV persistence and stability, and pgrna, an indicator of viral replication, correlate with viral activity and the phase of HBV infection. The combined measurement of cccdna and pgrna levels may provide valuable information on HBV natural history and the efficacy of long-term antiviral therapy by monitoring the persistence and replicative activity of both wild type and mutant HBV strains. Acknowledgment: The authors thank Dr. Peter Karayiannis for helpful discussions and critical reading of the manuscript. References 1. Hadziyannis SJ. Hepatitis B e antigen negative chronic hepatitis B: from clinical recognition to pathogenesis and treatment. Viral Hepat Rev 1995; 1: Lee WM. Hepatitis B virus infection. N Engl J Med 1997;337: Lok AS, McMahon BJ. Chronic hepatitis B. HEPATOLOGY 2001;34: Nassal M, Schaller H. Hepatitis B virus replication an update. J Viral Hepat 1996;3: Seeger C, Mason WS. Hepatitis B virus biology. Microbiol Mol Biol Rev 2000;64: Summers J, Mason WS. Replication of the genome of a hepatitis B like virus by reverse transcription of an RNA intermediate. Cell 1982;29: Bock CT, Schranz P, Schroder CH, Zentgraf H. Hepatitis B virus genome is organized into nucleosomes in the nucleus of the infected cell. Virus Genes 1994;8: Newbold JE, Xin H, Tencza M, Sherman G, Dean J, Bowden S, et al. The covalently closed duplex form of the hepadnavirus genome exists in situ as a heterogeneous population of viral minichromosomes. J Virol 1995;69: Tuttleman JS, Pourcel C, Summers J. Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell 1986;47: Kramvis A, Kew MC. The core promoter of hepatitis B virus. J Viral Hepat 1999;6: Yaginuma K, Shirakata Y, Kobayashi M, Koike K. Hepatitis B virus (HBV) particles are produced in a cell culture system by transient expression of transfected HBV DNA. Proc Natl Acad Sci USA1987;84: Will H, Reiser W, Weimer T, Pfaff E, Buscher M, Sprengel R, et al. Replication strategy of human hepatitis B virus. J Virol 1987;61: Takahashi K, Kishimoto S, Ohori K, Yoshizawa H, Machida A, Ohnuma H, et al. Molecular heterogeneity of e antigen polypeptides in sera from carriers of hepatitis B virus. J Immunol 1991;147: Wu TT, Coates L, Aldrich CE, Summers J, Mason WS. In hepatocytes infected with duck hepatitis B virus, the template for viral RNA synthesis is amplified by an intracellular pathway. Virology 1990;175: Carman WF, Jacyna MR, Hadziyannis S, Karayiannis P, McGarvey MJ, Makris A, et al. Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet 1989;2: Gunther S, Fischer L, Pult I, Sterneck M, Will H. Naturally occurring variants of hepatitis B virus. Adv Virus Res 1999;52: Hunt CM, McGill JM, Allen MI, Condreay LD. Clinical relevance of hepatitis B viral mutations. HEPATOLOGY 2000;31: Funk ML, Rosenberg DM, Lok AS. World-wide epidemiology of HBeAgnegative chronic hepatitis B and associated pre-core and core promoter variants. J Viral Hepat 2002;9: Hadziyannis SJ, Vassilopoulos D. Hepatitis B e antigen-negative chronic hepatitis B. HEPATOLOGY 2001;34: Buckwold VE, Xu Z, Chen M, Yen TS, Ou JH. Effects of a naturally occurring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication. J Virol 1996;70: Gunther S, Piwon N, Will H. Wild-type levels of pregenomic RNA and replication but reduced pre-c RNA and e-antigen synthesis of hepatitis B virus with C(1653) 3 T, A(1762) 3 T and G(1764) 3 A mutations in the core promoter. J Gen Virol 1998;79(Pt 2): Laras A, Koskinas J, Hadziyannis SJ. In vivo suppression of precore mrna synthesis is associated with mutations in the hepatitis B virus core promoter. Virology 2002;295: Moriyama K, Okamoto H, Tsuda F, Mayumi M. Reduced precore transcription and enhanced core-pregenome transcription of hepatitis B virus DNA after replacement of the precore-core promoter with sequences associated with e antigen-seronegative persistent infections. Virology 1996; 226: Yokosuka O, Omata M, Imazeki F, Okuda K, Summers J. Changes of hepatitis B virus DNA in liver and serum caused by recombinant leukocyte interferon treatment: analysis of intrahepatic replicative hepatitis B virus DNA. HEPATOLOGY 1985;5: Mason WS, Cullen J, Saputelli J, Wu TT, Liu C, London WT, et al. Characterization of the antiviral effects of 2 carbodeoxyguanosine in ducks chronically infected with duck hepatitis B virus. HEPATOLOGY 1994; 19: Luscombe C, Pedersen J, Uren E, Locarnini S. Long-term ganciclovir chemotherapy for congenital duck hepatitis B virus infection in vivo: effect on intrahepatic-viral DNA, RNA, and protein expression. HEPATOLOGY 1996;24: Chen RY, Edwards R, Shaw T, Colledge D, Delaney WE, Isom H, et al. Effect of the G1896A precore mutation on drug sensitivity and replication yield of lamivudine-resistant HBV in vitro. HEPATOLOGY 2003;37: Tacke F, Gehrke C, Luedde T, Heim A, Manns MP, Trautwein C. Basal core promoter and precore mutations in the hepatitis B virus genome

9 702 LARAS ET AL. HEPATOLOGY, September 2006 enhance replication efficacy of Lamivudine-resistant mutants. J Virol 2004;78: Addison WR, Wong WW, Fischer KP, Tyrrell DL. A quantitative competitive PCR assay for the covalently closed circular form of the duck hepatitis B virus. Antiviral Res 2000;48: He ML, Wu J, Chen Y, Lin MC, Lau GK, Kung HF. A new and sensitive method for the quantification of HBV cccdna by real-time PCR. Biochem Biophys Res Commun 2002;295: Jun-Bin S, Zhi C, Wei-Qin N, Jun F. A quantitative method to detect HBV cccdna by chimeric primer and real-time polymerase chain reaction. J Virol Methods 2003;112: Singh M, Dicaire A, Wakil AE, Luscombe C, Sacks SL. Quantitation of hepatitis B virus (HBV) covalently closed circular DNA (cccdna) in the liver of HBV-infected patients by LightCycler real-time PCR. J Virol Methods 2004;118: Wong DK, Yuen MF, Yuan H, Sum SS, Hui CK, Hall J, et al. Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients. HEPATOLOGY 2004;40: Werle-Lapostolle B, Bowden S, Locarnini S, Wursthorn K, Petersen J, Lau G, et al. Persistence of cccdna during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy. Gastroenterology 2004;126: Sung JJ, Wong ML, Bowden S, Liew CT, Hui AY, Wong VW, et al. Intrahepatic hepatitis B virus covalently closed circular DNA can be a predictor of sustained response to therapy. Gastroenterology 2005;128: Addison WR, Walters KA, Wong WW, Wilson JS, Madej D, Jewell LD, et al. Half-life of the duck hepatitis B virus covalently closed circular DNA pool in vivo following inhibition of viral replication. J Virol 2002;76: Wieland SF, Spangenberg HC, Thimme R, Purcell RH, Chisari FV. Expansion and contraction of the hepatitis B virus transcriptional template in infected chimpanzees. Proc Natl Acad Sci USA2004;101: Zhang YY, Zhang BH, Theele D, Litwin S, Toll E, Summers J. Single-cell analysis of covalently closed circular DNA copy numbers in a hepadnavirus-infected liver. Proc Natl Acad Sci USA2003;100: Zhu Y, Yamamoto T, Cullen J, Saputelli J, Aldrich CE, Miller DS, et al. Kinetics of hepadnavirus loss from the liver during inhibition of viral DNA synthesis. J Virol 2001;75: Ishak K, Baptista A, Bianchi L, Callea F, De Groote J, Gudat F, et al. Histological grading and staging of chronic hepatitis. J Hepatol 1995;22: Su Q, Wang SF, Chang TE, Breitkreutz R, Hennig H, Takegoshi K, et al. Circulating hepatitis B virus nucleic acids in chronic infection: representation of differently polyadenylated viral transcripts during progression to nonreplicative stages. Clin Cancer Res 2001;7: Zhang W, Hacker HJ, Tokus M, Bock T, Schroder CH. Patterns of circulating hepatitis B virus serum nucleic acids during lamivudine therapy. J Med Virol 2003;71: Summers J, Smith PM, Horwich AL. Hepadnavirus envelope proteins regulate covalently closed circular DNA amplification. J Virol 1990;64: Lenhoff RJ, Summers J. Construction of avian hepadnavirus variants with enhanced replication and cytopathicity in primary hepatocytes. J Virol 1994;68: