Hepatitis B Virus Basal Core Promoter Mutation and DNA Load Correlate with Expression of Hepatitis B Core Antigen in Patients with Chronic Hepatitis B

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1 MAJOR ARTICLE Hepatitis B Virus Basal Core Promoter Mutation and DNA Load Correlate with Expression of Hepatitis B Core Antigen in Patients with Chronic Hepatitis B Chun-Jen Liu, 1,2,a Yung-Ming Jeng, 3 Chi-Lin Chen, 1 Huei-Ru Cheng, 1 Pei-Jer Chen, 1,2,4, Ting-Chi Chen, 1 Chen-Hua Liu, 2 Ming-Yang Lai, 1,2 Ding-Shinn Chen, 1,2,5 and Jia-Horng Kao 1,2,4,5,a 1 Graduate Institute of Clinical Medicine, Departments of 2 Internal Medicine, 3 Pathology, and 4 Medical Research, and 5 Hepatitis Research Center, National Taiwan University College of Medicine and National Taiwan University Hospital, Taipei Background. Expression of intrahepatic hepatitis B core antigen (HBcAg) is related to the immunopathogenesis of hepatitis B virus (HBV) infection. This study investigated the role that HBV genotype and basal core promoter (BCP) mutation play in the expression of HBcAg. Methods. A total of 70 hepatitis B e antigen (HBeAg) positive patients with chronic hepatitis (genotype B in 52 patients and genotype C in 18 patients; BCP mutation T1762/A1764 in 16 patients) were enrolled. Clinical, virologic, and histologic features were compared with regard to localization and expression of intrahepatic HBcAg. The effects that HBV genotype and BCP mutation T1762/A1764 had on expression of HBcAg were further evaluated by in vitro assays. Results. Cytoplasmic, mixed cytoplasmic/nuclear, and nuclear localization of intrahepatic HBcAg was found in 38 (56.7%), 25 (37.3%), and 4 (6.0%) patients, respectively; HBcAg was not discernible in 3 patients. A total of 58 (82.9%) of these patients expressed a high level of HBcAg. In multivariate analysis, cytoplasmic localization of HBcAg correlated only with a low HBV load in serum (P.045) and BCP mutation (P.04). A high expression level of HBcAg also correlated with a high HBV load in serum (P.015) and with BCP wild-type sequence (P.037). In vitro assays indicated that the HBV BCP mutant strain had lower subcellular expression of HBcAg than did the BCP wild-type strain. Conclusions. HBV BCP mutation and HBV load, but not genotype, contribute to the expression of intrahepatic HBcAg. Chronic hepatitis B virus (HBV) infection is a major health problem worldwide [1]. The pathogenesis of Received 22 July 2008; accepted 19 September 2008; electronically published 26 January Potential conflicts of interest: none reported. Financial support: National Taiwan University Hospital (grant NTUH-94A06); National Science Council, Taiwan (grants NSC B , NSC B , NSC B , NSC B MY3, and NSC B ); Department of Health, Executive Yuan, Taiwan; National Taiwan University (grant 95R0066-BM02-04); and National Health Research Institutes, Taiwan. a C.-J.L and J.-H.K. contributed equally to this work. Reprints or correspondence: Prof. Jia-Horng Kao, Director and Distinguished Professor, Hepatitis Research Center, National Taiwan University Hospital, 1 Chang-Te St., Taipei 10002, Taiwan (kaojh@ntu.edu.tw); or Dr. Chun-Jen Liu, Assoc. Prof., Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, 1 Chang-Te Street, Taipei 10002, Taiwan (cjliu@ntu.edu.tw). The Journal of Infectious Diseases 2009; 199: by the Infectious Diseases Society of America. All rights reserved /2009/ $15.00 DOI: / HBV infection is mediated mainly via host immune responses to hepatocytes presenting HBV-encoded antigens, particularly hepatitis B core antigen (HBcAg) [2]. HBcAg, a translational product of the precore/core gene, is a protein required for the packaging of viral DNA into core particles, which is a critical process in the life-cycle of HBV. The existence of HBcAg in the liver is therefore a hallmark of active HBV replication and is closely associated with the presence of hepatitis B e antigen (HBeAg) and high serum levels of HBV DNA. In the natural course of perinatally acquired HBV infection, predominantly cytoplasmic expression of HBcAg is usually found during the immune-clearance phase, when, on the basis of histological analysis of the liver, patients have episodes of hepatitis flare and active hepatitis; in contrast, HBcAg is predominantly distributed in the nucleus during the immune-tolerance phase, when pa- 742 JID 2009:199 (1 March) Liu et al.

2 tients have livers that either are normal or have only minimal injury [3, 4]. Although subcellular localization of HBcAg is related to the viral replication and disease activity of chronic HBV infection, factors affecting their distribution and expression remain largely unknown. Previous studies have suggested that HBV load and severity of hepatocyte injury and subsequent hepatocyte regeneration might influence both the pattern and the level of HBcAg expression in the liver [5 10]. Recently, HBV genotypes and mutant strains have been shown to affect the natural course and treatment outcomes of chronic HBV infection [11 32]; for example, mutations in the precore and the basal core promoter (BCP) genes may be associated with the progression of liver disease [11 32]. However, the molecular and virologic mechanisms accounting for such a genotype-phenotype relationship are far from clear and therefore require further investigation [19]. Because intracellular localization of HBcAg is closely related to the underlying immunopathogenesis and disease activity of HBV infection [6 10], whether there is any association between HBV genotypes or mutant strains and subcellular localization of HBcAg remains an interesting and important issue. We hypothesized that HBV genotypes or naturally occurring mutant strains may affect the localization and expression of intrahepatic HBcAg, which then influence the presentation of HBcAg and subsequent immune-mediated hepatocytolysis. Indeed, recent studies have revealed that cytoplasmic localization of HBcAg is associated with mutations of either core promoter nucleotides 1762 and 1764 or precore nucleotide 1896 [33 35]. Another study also has demonstrated that high HBV DNA levels and genotype C infection, rather than core-promoter or precore stopcodon mutations, are associated with more-severe liver damage in HBeAg-negative carriers [36]. However, the populations in these studies were heterogeneous, and in vitro evidence was still lacking. To address this issue, we consecutively enrolled a homogeneous population of HBV carriers who were HBeAg positive and highly viremic and who had histologically proven active hepatitis. The relationship between HBV genotype, BCP mutation, and subcellular localization/expression of HBcAg in the liver was further clarified. Additionally, the effects that HBV genotype and BCP mutation T1762/A1764 have on the expression of HBcAg were examined by in vitro mutagenesis and transfection assays. PATIENTS AND METHODS Patients. From October 2004 through December 2007, 70 patients with chronic hepatitis B which was defined as the presence of hepatitis B surface antigen (HBsAg) in serum for 6 months and elevated serum levels ( 2 times the upper limit of normal on 2 occasions during the preceding 6 months) of alanine aminotransferase were consecutively enrolled. The liverbiopsy results of all patients were compatible with the diagnosis of chronic liver disease, and all patients were positive for HBeAg and had not received interferon, lamivudine, other experimental antiviral agents, or immunosuppressive therapy before being enrolled in the study. The patients had no serologic evidence of autoimmune liver disease or inheritable disorders such as hemochromatosis or Wilson disease or any history of alcoholism or drug abuse, and none of them had concomitant HIV, hepatitis C virus (HCV), or hepatitis D virus (HDV) infection. After written informed consent was obtained, serum and liver-tissue samples were collected simultaneously and were stored at 70 C until assayed. Serological assays, HBV genotyping, and quantification of HBV DNA in serum. Hepatitis markers were assayed by use of commercially available kits (Ausab, Ausria II, Anti-HCV, and Anti-HDV; Abbott Laboratories). The serum samples were also tested for HBeAg and anti-hbe, by use of enzyme-immunoassay kits (Cobas-Core; Roche). Genotyping of HBV and quantification of serum levels of HBV DNA were performed as described elsewhere [37, 38]. Analysis of precore and BCP genes. The presence of precore and BCP mutant strains was detected as described elsewhere [20]. In brief, the precore and BCP regions of HBV were amplified by nested-pcr assays. The PCR products were then directly sequenced by use of an automatic ABI DNA sequencer (Model 377A; Applied Biosystems) to determine the nucleotide sequences, with emphasis on nucleotides 1762, 1764, and Effects that HBV genotype and BCP mutations have on intracellular expression of HBcAg. We compared the intracellular expression of HBcAg in the BCP wild-type strain versus that in the BCP mutant strain by using in vitro mutagenesis to create HBV strains carrying the candidate BCP mutation T1762/ A1764. The location of HBcAg was determined by immunofluorescence assay, and its expression level was determined by Western blot assay. Cloning of PCR products, isolation of plasmid DNA, and preparation of full-length HBV genomes for mutagenesis and transfection. These procedures were performed as previously elsewhere [37, 38]. Preparation of site-directed BCP T1762/A1764 mutant strains. Mutations in the BCP region were introduced by overlap extension PCR using the Expand high-fidelity PCR system (PfuUltra high-fidelity [HF] DNA polymerase; Stratagene) for 25 cycles. The PCR products were digested with restriction enzymes RsrII and ApaI to exchange them with the cognate fragment in suitable clones. The entire PCR-derived fragment was sequenced to confirm that there were no other mutations. Cell culture and transfection. Huh7 cells were maintained in 5% CO 2 in Dulbecco s modified Eagle medium supplemented with 10% fetal bovine serum, penicillin at 50 U/mL, streptomycin at 50 U/mL, glutamine at 1 mmol/l, and 10 mol of nonessential amino acids. The cells were plated on a 3.5-cm-diameter petri dish and were incubated for 24 h; 1 g of pegfp-n1 vector HBV Factors and Intrahepatic HBcAg Expression JID 2009:199 (1 March) 743

3 encoding green fluorescent protein (Clontech Laboratories) was cotransfected as an internal control to monitor transfection efficiency; and 2 g of HBV DNA was used for transfection into Huh7 cells, by use of Arrest-In transfection reagent (Arrest-In transfection reagent for RNAi; Open Biosystems), according to the manufacturers instructions. The medium was replaced with fresh medium both 24 h and 3 days after transfection. The cells and the final medium were harvested 5 days after transfection. All experiments were performed 5 times. Immunofluorescence assay. For immunofluorescent staining, cells were cultured on uncoated glass cover slips. At 3 days after transfection, the cover slips were rinsed 3 times with PBS and then were fixed with 4% paraformaldehyde (Paraformaldehyde power 95%; Sigma-Aldrich) in PBS at room temperature for 10 min, were permeabilized with 0.2% (vol/vol) Triton X-100 (Sigma-Aldrich) in PBS on ice for 20 min, and were incubated in 4% blocking buffer (4% bovine serum albumin [BSA]/ PBS) for1hat37 C. The cells then were incubated sequentially with primary and secondary antibodies. Intrahepatic HBcAg was probed, overnight at 4 C, by use of rabbit anti-hbc (Polyclonal Rabbit Anti-Hepatitis B Core Antigen; Dako) diluted in 1% BSA/PBS (1:1200). The slides were washed 3 times for 5 min with PBS and then were probed, by use of secondary anti-rabbit antibody (Anti-Rabbit IgG, H& LChain Specific [Goat] Fluorescein Conjugate; EMD Biosciences) diluted in 1% BSA/PBS (1: 1200), for 1hatroom temperature. The slides then were washed 3 times for 5 min with PBS. After being immunostained, the cover slips were mounted, overnight at 4 C, on slides in 3 5 L of mounting medium with 4',6'-diamidino-2-phenylindole (Vectashield Hard Set Mounting Medium with DAPI; Vector Laboratories). The slide samples were read by use of a fluorescence microscope. Determination of amount of intrahepatic core antigen by Western blot assay. We determined the expression level of intrahepatic HBcAg in comparison to that of -actin by using a Western blot assay. Histologic evaluation. All patients livers were histologically graded and staged by use of Knodell s scoring system, which takes into account the severity of periportal, portal, and lobular necroinflammation and fibrosis [39]. The localization and expression level of HBcAg in the hepatocytes were analyzed after immunohistochemical staining, as described elsewhere [40]. In brief, the expression pattern of HBcAg was classified as either predominantly nuclear, mixed nuclear/cytoplasmic, or predominantly cytoplasmic. The expression level of HBcAg in a 200 high-power field was arbitrarily categorized as either low ( 30 hepatocytes stained with HBcAg) or high ( 30 hepatocytes stained with HBcAg). At least 2 representative views of each case were counted and averaged. A single pathologist (Y.-M.J) who was blinded to clinical information reviewed all of the liverbiopsy samples simultaneously. Table 1. Baseline characteristics of 70 patients with hepatitis B e antigen positive chronic hepatitis B. Characteristic Ethical considerations. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Ethical Committee of the National Taiwan University Hospital (NTUH ). Statistical analysis. All categorical and continuous variables were analyzed by 2 test and Student s t test, respectively. For continuous variables with outliers, nonparametric tests were used. Possible factors associated with the subcellular localization or expression level of HBcAg in the liver were assessed by univariate and multivariate analyses. P.05 was considered to be statistically significant. RESULTS Value Males, no. (%) 58 (82.7) Age, mean SD, years ALT, mean SD, IU/L Serum level of HBV DNA, mean SD, log 10 copies/ml HBV genotype: B/C, no. (%) 52/18 (74.3/25.7) Precore: G1896/A1896/undetectable, no. (%) 52/15/3 (74.3/21.4/4.3) BCP: A1762/G1764 vs. T1762/A1764 vs. undetectable, no. (%) 48/16/6 (68.6/22.9/8.5) Hepatitis activity index, mean SD Fibrosis score, mean SD NOTE. ALT, alanine aminotransferase; BCP, basal core promoter; HBV, hepatitis B virus. Patients. The clinical features of the 70 HBeAg-positive patients with chronic active hepatitis are shown in table 1. Overall, they were relatively young and highly viremic, and most were males. Distribution pattern and expression level of intrahepatic HBcAg, and their correlation with HBV genotype and BCP mutations. The distribution pattern and expression level of HBcAg in the liver are shown in tables 2 and 3. Most of the samples had predominantly cytoplasmic or mixed cytoplasmic/ nuclear localization of HBcAg and expressed high levels of intrahepatic HBcAg. When clinical, histologic, and virologic features were compared over the cytoplasmic, nuclear, and mixed cytoplasmic/ nuclear localization of intrahepatic HBcAg, only BCP mutation and a low serum level of HBV DNA were found to be correlated with cytoplasmic localization of HBcAg, both in univariate analysis and in multivariate analysis (tables 2 and 4). When similar comparisons were made between high and low expression levels of HBcAg, the BCP A1762/G1764 wild-type sequence and a high serum level of HBV DNA were found to be significantly associated with a high expression level of intrahe- 744 JID 2009:199 (1 March) Liu et al.

4 Table 2. Profiles of patients with different distribution patterns of intrahepatic hepatitis B core antigen (HBcAg). Distribution pattern Category Cytoplasmic Mixed Nuclear Cases, no. (%) 38 (56.7) 25 (37.3) 4 (6.0)... Males/females, no. (%) 31/7 (81.6/18.4) 21/4 (84.0/16.0) 4/0 (100/0).638 Age, mean SD, years ALT, mean SD, IU/L Serum level of HBV DNA, mean SD, log 10 copies/ml HBV genotype: B/C, no. (%) 28/10 (73.7/26.3) 19/6 (76.0/24.0) 3/1 (75.0/25.0).979 Precore: G1896/A1896, no. (%) 28/10 (73.7/26.3) 20/5 (80.0/20.0) 3/0 (100/0).531 BCP: A1762/G1764 vs. T1762/A1764, no. (%) 24/12 (66.7/33.3) 22/2 (91.7/8.3) 1/2 (33.3/66.7).023 Hepatitis activity index, mean SD Fibrosis score, mean SD NOTE. All categorical and continuous variables were analyzed by 2 test and Student s t test, respectively; for continuous variables with outliers, nonparametric tests were used. ALT, alanine aminotransferase; BCP, basal core promoter; HBV, hepatitis B virus. a Because of the limited no. of such cases, patients with predominantly nuclear localization of HBcAg were not included in the comparisons. Significant values are in boldface type. HBcAg was not discernible in 3 patients. Table 3. (HBcAg). Category patic HBcAg (table 3). Multivariate analysis found that the BCP A1762/G1764 wild-type sequence and a high serum level of HBV DNA continued to be significantly associated with a high expression level of HBcAg (table 5). Factors correlated with the stage of hepatic fibrosis. We also investigated the factors correlated with the stage of hepatic fibrosis in patients with chronic hepatitis B; we found that only a high HBV load in serum and a high hepatitis-activity index were positively correlated with the presence of advanced fibrosis in the liver-biopsy samples (data not shown). Effect that HBV genotype and BCP mutations have on the location and expression of intrahepatic HBcAg in vitro. To further investigate the effect that HBV genotype and BCP mutations have on the expression of intracellular HBcAg, we selected 1 patient with HBV genotype B and 1 patient with HBV genotype C who were infected with the BCP A1762/G1764 wild-type strain. After in vitro mutagenesis, these strains were documented as exhibiting BCP mutation T1762/A1764, a result that was confirmed by direct sequencing. After transfection, the expression of intracellular HBcAg was found, by flow cytometry, to be localized primarily in the cytoplasm, regardless of HBV genotype or BCP dinucleotide status (figure 1). Using Western blot analysis, we demonstrated that the expression level of intracellular HBcAg was lower in the BCP T1762/A1764 mutant strain than in the A1762/G1764 wild-type strain, in both genotype B and genotype C (figure 2). Profiles of patients with different levels of expression of intrahepatic hepatitis B core antigen Expression level of HBcAg a Cases, no. (%) 58 (82.9) 12 (17.1)... Males, no. (%) 47 (81.0) 11 (91.7).676 Age, mean SD, years ALT, mean SD, IU/L Serum level of HBV DNA, mean SD, log 10 copies/ml <.001 HBV genotype: B/C, no. (%) 44/14 (75.9/24.1) 8/4 (66.7/33.3).49 Precore: G1896/A1896, no. (%) 43/15 (74.1/25.9) 9/0 (100/0).191 BCP: A1762/G1764 vs. T1762/A1764, no. (%) 45/11 (80.4/19.6) 3/5 (37.5/62.5).019 Hepatitis activity index, mean SD Fibrosis score, mean SD High Low P b P a NOTE. All categorical and continuous variables were analyzed by 2 test and Student s t test, respectively; for continuous variables with outliers, nonparametric tests were used. ALT, alanine aminotransferase; BCP, basal core promoter; HBV, hepatitis B virus. a Low, 30 hepatocytes stained with HBcAg under 200 high-power field; high, 30 hepatocytes. b Significant values are in boldface type. HBV Factors and Intrahepatic HBcAg Expression JID 2009:199 (1 March) 745

5 Table 4. Multivariate analysis of factors associated with cytoplasmic localization of intrahepatic hepatitis B core antigen. Category OR (95% CI) P a Age, years ( ).636 Sex, females:males ( ).236 HBV DNA, log 10 copies/ml ( ).045 Fibrosis score ( ).585 Hepatitis activity index ( ).515 Precore, A1896:G ( ).315 BCP, T1762/A1764:A1762/G ( ).04 Genotype, C:B ( ).464 NOTE. BCP, basal core promoter; CI, confidence interval; HBV, hepatitis B virus; OR, odds ratio. a Significant values are in boldface type. DISCUSSION The present study investigated the association between the genomic variability of HBV strains and the expression of intrahepatic HBcAg in HBeAg-positive patients with chronic hepatitis B. The clinical results revealed that subcellular cytoplasmic localization and a low expression level of HBcAg correlate with BCP mutation T1762/A1764 and with a lower serum level of HBV DNA. In vitro studies confirmed that the low expression level of subcellular HBcAg is correlated with the BCP mutant sequence. Previous studies have demonstrated that dinucleotide mutation A1762T/G1764A in BCP affects codons 130 and 131 of the HBx gene (K130M and V131I) and that this dinucleotide mutation is correlated with decreased expression of HBeAg and with enhanced viral replication [14, 25, 29, 33, 40]. Previous association and cohort studies also have indicated that HBV genotype C and the BCP T1762/A1764 mutant strain correlate with poor clinical outcomes, compared with genotype B and the BCP A1762/G1764 wild-type strain, respectively [16 24]. However, only a few in vitro studies have examined the biological differences between either HBV genotypes or BCP strains. In the present study, we first provided in vivo evidence to support the notion that HBV BCP mutation T1762/A1764 is significantly associated with cytoplasmic localization of intracellular HBcAg. Consistent with our findings, the results of another recent study have demonstrated that HBcAg s cytoplasmic localization is more associated with BCP T1762/A1764 than is its nuclear expression [34]. Because cytoplasmic localization of HBcAg is closely related to active necroinflammation of hepatocytes [41], these findings may partly explain why the emergence of the BCP mutant strain is associated with worse clinical outcomes than is the BCP wild-type strain. Nevertheless, at the molecular level, mechanisms accounting for the migration and localization of intracellular HBcAg remain unclear, although phosphorylationdependent binding of hepatitis B core particles to the nuclear pore complex, possibly in the serine residues of the SPRRR motif of the hepatitis B core gene, has been proposed [42 45]. Whether nuclear localization of HBcAg is due to decreased migration of HBcAg and whether BCP T1762/A1764 mutant strains have any impact on the phosphorylation-dependent binding of the hepatitis B core particles to the nuclear pore complex remains unknown. Future studies should focus on the immunopathogenic roles played by these BCP mutations and the related, altered HBx protein. The present study has found that BCP A1762/G1764 wildtype strains, rather than BCP T1762/A1764 mutant strains, are significantly associated with a high level of expression of intrahepatic HBcAg. On the basis of these data on HBeAg-positive patients with chronic hepatitis B during the immune-clearance phase, we propose that a BCP strain dependent difference in the expression of HBV antigens exists during the earlier stages of chronic HBV infection. Nevertheless, in a previous study, BCP mutant strains have been shown to possess enhanced viral replication [25]. Mechanisms accounting for these discrepant results in both viral replication and HBcAg expression in BCP mutant strains await further molecular and virologic analyses. The role that HBV genotype and BCP mutant strains play in HBeAg-negative chronic hepatitis B, a later reactivation phase of chronic HBV infection, also needs to be studied. HBV genotype has been shown to correlate with clinical and treatment outcomes [46]. A recent study has also shown that the expression of intrahepatic HBsAg is significantly greater in patients infected with genotype C than in those infected with genotype B [47]. In the present study, we have demonstrated that HBV genotype is not associated with the location or expression level of intrahepatic HBcAg. Accordingly, the mechanisms accounting for the clinical manifestations of various HBV genotypes remain to be clarified. The present study had limitations that might result in ambiguous inferences with regard to the relationship between the localization and expression of intrahepatic HBcAg and the presence of HBV mutant strains. First, we considered only an HBV precore stop-codon mutation (A1896), a dinucleotide mutation (T1762/A1764) in the basal core promoter, and genotype in Table 5. Multivariate analysis of factors associated with high levels of expression of intrahepatic hepatitis B core antigen. Category OR (95% CI) P a Age, years ( ).971 Sex, females:males ( ).445 HBV DNA, Log 10 copies/ml ( ).015 Fibrosis, score ( ).469 Hepatitis activity index, score ( ).514 BCP, T1762/A1764:A1762/G ( ).037 Genotype, C: B ( ).746 NOTE. BCP, basal core promoter; CI, confidence interval; HBV, hepatitis B virus; OR, odds ratio. a Significant values are in boldface type. 746 JID 2009:199 (1 March) Liu et al.

6 Figure 1. Expression of intracellular hepatitis B core antigen in Huh 7 cells transfected with HBV genotype B isolated from case clones carrying the A1762/G1764 wild-type sequence before (A) and after (C) in vitro mutagenesis and in Huh 7 cells transfected with HBV genotype C isolated from case clones carrying the T1762/A1764 mutant sequence before (B) and after (D) in vitro mutagenesis. HBeAg-positive patients with active hepatitis. It has been speculated that the clinical phenotypes of HBV infection may be determined by multiple point mutations along the HBV genomes [48], and therefore the possibility that other viral mutant strains contribute to the localization and expression of intrahepatic HBcAg cannot be excluded. Second, the HBV genome Figure 2. Intrahepatic hepatitis B core antigen (HBcAg) quantified by Western blot assay: comparison between hepatitis B virus (HBV) wildtype and mutant basal core promoter (BCP). Shown are mean SD values of intrahepatic HBcAg obtained after summation of 5 independent assays, expressed in terms of the ratio of HBcAg to -actin, evolves over time, and, in a cross-sectional study, we could not clarify the causal relationship between the localization/expression of intrahepatic HBcAg and HBV mutant strains. Thus, prospective studies are necessary to assess the temporal relationships among viral genomic evolution, viral dynamics, hepatitis activity, and localization/expression of intrahepatic HBcAg. Third, in our in vitro assay, we could not demonstrate the differential distribution of subcellular HBcAg after short-term transfection. To address this issue, the use of alternative cell lines or strategies should be considered. Fourth, caution should be exercised in extrapolating the results of our in vitro experiments to clinical scenarios, because the duration of infection or host immune response was not taken into account in the present study. Fifth, apart from viral factors, host factors should also be considered in analyses of the intrahepatic expression of viral antigens [49]. Finally, for statistical analysis, the expression level of HBcAg in a 200 high-power field was arbitrarily considered to be 30 hepatocytes stained with HBcAg versus 30 hepatocytes stained with HBcAg; however, when the cutoff expression level of HBcAg was set at 50 or 100 hepatocytes stained with HBcAg, similar results were obtained (data not shown). In summary, the results of the present study suggest that BCP mutation T1762/A1764 and HBV load contribute to the expres- HBV Factors and Intrahepatic HBcAg Expression JID 2009:199 (1 March) 747

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