Differential roles of serum hepatitis B virus DNA and hepatitis B surface antigen level in predicting virological breakthrough in patients receiving lamivudine therapy.

BACKGROUND AND AIM: The role of serum hepatitis B surface antigen (HBsAg) level in determining virological breakthrough (VB) for patients with hepatitis B virus (HBV) infection receiving lamivudine remains unclear. The study aimed to evaluate the impact of serum HBsAg levels on VB among patients receiving lamivudine therapy, especially in a setting of low HBV viral load. METHODS: Two hundred sixty-eight consecutive treatment-naïve patients who underwent lamivudine therapy for chronic hepatitis B were enrolled. Factors in terms of VB were analyzed by multivariate analysis. RESULTS: After a median treatment duration of 67.1 weeks, 102 patients had VB. Multivariate analysis showed that positive hepatitis B e antigen (HBeAg) (hazard ratio 2.165, P = 0.026) and HBV DNA levels ≥ 2000 IU/mL after 6 months of lamivudine therapy (hazard ratio 5.236, P = 0.001) were independent risk factors predicting VB. The cumulative VB rates stratified by HBeAg-positive and -negative at 3 years were 44.7% and 26.3%, respectively. At 3 years, the cumulative VB rates stratified by the HBV DNA < 2000 and ≥ 2000 IU/mL after 6 months of therapy were 25.5% and 79.4%, respectively. For HBeAg-positive patients with serum HBV DNA < 2000 IU/mL after 6 months of therapy, baseline HBsAg levels ≥ 20,000 IU/mL was the only risk factor associated with VB. CONCLUSIONS: For chronic hepatitis B patients treated with lamivudine, serum HBV DNA level > 2000 IU/mL after 6 months of therapy could predict subsequent VB. In patients with lower on-treatment viral load, baseline serum HBsAg level is associated with the emergence of VB, especially for those with serum positive HBeAg.

Hepatitis D virus isolates with low replication and epithelial-mesenchymal transition-inducing activity are associated with disease remission.

Clearance of hepatitis D virus (HDV) viremia leads to disease remission. Large hepatitis delta antigen (L-HDAg) has been reported to activate transforming growth factor ß, which may induce epithelial-mesenchymal transition (EMT) and fibrogenesis. This study analyzed serum HDV RNA "quasispecies" in HDV-infected patients at two stages of infection: before and after alanine aminotransferase (ALT) elevations. Included in the study were four patients who went into remission after ALT elevation and three patients who did not go into remission and progressed to cirrhosis or hepatocellular carcinoma. Full-length HDV cDNA clones were obtained from the most abundant HDV RNA species at the pre- and post-ALT elevation stages. Using an in vitro model consisting of Huh-7 cells transfected with cloned HDV cDNAs, the pre- or post-ALT elevation dominant HDV RNA species were characterized for (i) their replication capacity by measuring HDV RNA and HDAg levels in transfected cells and (ii) their capacity to induce EMT by measuring the levels of the mesenchymal-cell-specific protein vimentin, the EMT regulators twist and snail, and the epithelial-cell-specific protein E-cadherin. Results show that in patients in remission, the post-ALT elevation dominant HDV RNA species had a lower replication capacity in vitro and lower EMT activity than their pre-ALT elevation counterparts. This was not true of patients who did not go into remission. The expression of L-HDAg, but not small HDAg, increased the expression of the EMT-related proteins. It is concluded that in chronically infected patients, HDV quasispecies with a low replication capacity and low EMT activity are associated with disease remission.

Pro-205 of large hepatitis delta antigen and Pro-62 of major hepatitis B surface antigen influence the assembly of different genotypes of hepatitis D virus.

Hepatitis B surface antigen (HBsAg) is essential for the assembly and infection of hepatitis D virus (HDV). The assembly efficiency of genotype 1 HDV is higher than that of genotype 2, whilst the P62L substitution of major HBsAg further compromises the assembly of genotype 2 and 4 HDV. This study investigated the influence of proline residues in the carboxyl end of the large hepatitis delta antigen (HDAg-L) on the assembly of HDV of different genotypes. Expression vectors containing the HDAg-L gene or full-length HDV genome of genotype 1, 2 or 4 were co-transfected with plasmids expressing HBsAg proteins that bore either proline or leucine residues at position 62. Of the eight HDV genotypes, only genotype 1 has Pro-205 in HDAg-L, whereas genotypes 2 and 4 have Arg-205. The Arg-205 to Pro-205 substitution in HDV-2 and -4 markedly increased the assembly efficiencies of HDAg-L and whole HDV genomes, even in the presence of HBsAg with Leu-62. In contrast, secretion of genotype 1 HDV or HDAg-L was reduced significantly when arginine or alanine replaced Pro-205. When HBsAg contained Pro-62, the influence of Pro-205 on assembly decreased. In conclusion, both Pro-205 of the HDAg-L and Pro-62 of the major HBsAg play critical roles in the assembly of HDV of different genotypes. The presence of Pro-205 in genotype 1 HDV may account for its higher assembly efficiencies and wider distribution.

Risk factors for early and late recurrence in hepatitis B-related hepatocellular carcinoma.

BACKGROUND/AIMS: Hepatitis B virus (HBV) levels correlate with the development of hepatocellular carcinoma (HCC), but the role of viral load in HCC recurrence after tumor resection remains unclear. Herein we aimed to investigate the role of viral load in HCC recurrence following tumor resection. METHODS: From 1990 to 2002, 193 HBV-related HCC patients who underwent tumor resection in Taipei Veterans General Hospital were enrolled. Serum HBV DNA level and mutations were analyzed for association with early and late recurrence, together with other clinical variables. RESULTS: During a follow-up of 58.2+/-44 months, 134 patients had HCC recurrence. Multivariate analysis showed that multinodularity (Hazard ratio [HR], 95% confidence interval [CI]; 2.232, 1.021-4.878), macroscopic venous invasion (4.693, 1.645-13.391), AFP >20 ng/ml (3.891, 1.795-8.475), and cut margin 6 (4.658, 1.970-11.017), multinodularity (3.266, 1.417-7.526), ICG-15 >10% (2.487, 1.095-5.650) and HBV DNA level >10(6) copies/ml (2.548, 1.040-6.240) were significantly associated with late recurrence (>two years after resection). Patients with high viral loads tended to have higher Ishak inflammatory (7.00+/-3.07 vs. 5.33+/-2.96, p=0.001) and fibrosis scores (4.17+/-2.01 vs. 3.20+/-2.41, p=0.007) than those with lower loads. CONCLUSIONS: Tumor factors were associated with early HCC recurrence while high viral loads and hepatic inflammatory activity were associated with late recurrence. Pre- and post-operative antiviral and anti-inflammatory therapies may be crucial in reducing late recurrence.

Hepatitis B surface antigen levels and sequences of natural hepatitis B virus variants influence the assembly and secretion of hepatitis d virus.

Various domains of hepatitis B surface antigen (HBsAg) are essential for the assembly and secretion of hepatitis D virus (HDV). This study investigated the influences of the levels and sequences of HBsAg of naturally occurring HBV variants on the assembly and secretion of HDV. Six hepatitis B virus (HBV)-producing plasmids (three genotype B and three genotype C) and six HBsAg expression plasmids that expressed various HBsAg levels were constructed from the sera of HDV-infected patients. These plasmids were cotransfected with six expression plasmids of HDV of genotype 1, 2, or 4 into the Huh-7 hepatoma cell line. Serum HBsAg and HBV DNA levels were correlated with HDV RNA levels and outcomes of chronic hepatitis D (CHD) patients. The secretion of genotype 1, 2, or 4 HDV generally correlated with HBsAg levels but not with HBV genotypes or HBV DNA levels. Swapping and residue mutagenesis experiments of HBsAg-coding sequences revealed that the residue Pro-62 in the cytosolic domain-I affects the assembly and secretion of genotype 2 and 4 HDV and not those of genotype 1. The pre-S2 N-terminal deletion HBV mutant adversely affects secretion of the three HDV genotypes. In patients, serum HDV RNA levels correlated with HBsAg levels but not with HBV DNA levels. Viremia of HDV or HBV correlated with poor outcomes. In conclusion, the assembly and secretion of HDV were influenced by the amounts and sequences of HBsAg. For an effective treatment of CHD, reduction of HBsAg production in addition to the suppression of HBV and HDV replication might be crucial.

Positive selection of hepatitis delta antigen in chronic hepatitis D patients.

Liver disease may become ameliorated in some patients with chronic hepatitis D virus (HDV) infection. We present here a study based on longitudinal sampling to investigate the viral dynamics in chronic HDV infection. We examined the HDV variants from different time points, especially those before and after the elevation of serum aminotransferase levels. The datasets from each patient were tested for positive selection by using maximum-likelihood methods with heterogeneous selective pressures along the nucleotide sequence. An average of 4.9%, ranging from 3.1 to 6.8%, of the entire delta antigen sites was regulated by a diversifying selection. Most of the positively selected sites were associated with immunogenic domains. Likelihood ratio tests revealed a significant fitness of positive selection over neutrality of the hepatitis delta antigen gene in all patients. We further adapted a neural network method to predict potential cytotoxic T ligand epitopes. Among the HLA-A*0201 cytotoxic T ligand epitopes, three consistent epitopes across all three genotypes were identified: amino acids (aa) 43 to 51, 50 to 58, and 114 to 122. Three patients (60%) had sites evolving under positive selection in the epitope from aa 43 to 51, and four patients (80%) had sites evolving under positive selection in the epitope from aa 114 to 122. The discovery of immunogenic epitopes, especially cytotoxic-T-lymphocyte ligands, associated with chronic HDV infection may be crucial for further development of novel treatments or designs in vaccine for HDV superinfection.

Genotypes and viremia of hepatitis B and D viruses are associated with outcomes of chronic hepatitis D patients.

BACKGROUND & AIMS: Genotypes and viremia of hepatitis D virus (HDV) and hepatitis B virus (HBV) may be associated with outcomes. This study evaluated the impact of viral genotypes and viremia on outcomes of dual HBV and HDV infection. METHODS: Viremia and viral genotypes were analyzed in 194 consecutive chronic hepatitis B patients with HDV superinfection and correlated with outcomes. RESULTS: The numbers of HBV genotype A, B, C, and nonclassified were 4, 57, 23, and 110, respectively. There were 51 genotype I HDV, 74 genotype II HDV, 8 genotype IV HDV, and 61 nonclassified HDV genotype. In a median follow-up of 135 months, 24 progressed to cirrhosis and 41 developed hepatocellular carcinoma. Patients infected with genotype I HDV had a lower remission rate (15.2% vs 40.2%; P = .007) and more adverse outcomes (cirrhosis, hepatocellular carcinoma, or mortality) (52.2% vs 25.0%; P= .005) than those with genotype II HDV. Patients infected with genotype C HBV had a lower remission rate (0 vs 32.1%; P = .005) and more adverse outcomes (70.0% vs 33.9%; P = .005) than those with genotype B HBV. The presence of HBV or HDV viremia was associated with lower remission rates compared with those negative for both (26.4% and 24.3% vs 69.2%; P < .001). In multivariate analysis, age, genotype C HBV, and genotype I HDV were independent factors associated with adverse outcomes. CONCLUSIONS: In chronic HBV and HDV dual infections, older age, genotype I HDV, and genotype C HBV correlated with adverse outcomes.

"Defective" mutations of hepatitis D viruses in chronic hepatitis D patients.

AIM: To verify whether "defective" mutations existed in hepatitis D virus (HDV). METHODS: Hepatitis delta antigen (HDAg)-coding sequences were amplified using Pfu DNA polymerases with proof-reading activities from sera of five patients with chronic hepatitis D. Multiple colonies were sequenced for each patient. Pfu analyzed a total of 270 HDV clones. Three representative defective HDV clones were constructed in expression plasmids and transfected into a human hepatoma cell line. Cellular proteins were extracted and analyzed by Western blot. RESULTS: Four of five cases (80%) showed defective HDV genomes in their sera. The percentage of defective genomes was 3.7% (10/270). The majority (90%) of the defective mutations were insertions or deletions that resulted in frameshift and abnormal stop translation of the HDAg. The predicted mutated HDAg ranged from 45 amino acids to >214 amino acids in length. Various domains of HDAg associated with viral replication or packaging were affected in different HDV isolates. Western blot analysis showed defected HDAg in predicted positions. CONCLUSION: "Defective" viruses do exist in chronic HDV infected patients, but represented as minor strains. The clinical significance of the "defected" HDV needs further study to evaluate.

Interaction and replication activation of genotype I and II hepatitis delta antigens.

The nucleotide sequences of hepatitis D viruses (HDV) vary 5 to 14% among isolates of the same genotype and 23 to 34% among different genotypes. The only viral-genome-encoded antigen, hepatitis delta antigen (HDAg), has two forms that differ in size. The small HDAg (HDAg-S) trans-activates viral replication, while the large form (HDAg-L) is essential for viral assembly. Previously, it has been shown that the packaging efficiency of HDAg-L is higher for genotype I than for genotype II. In this study, the question of whether other functional properties of the HDAgs are affected by genotype differences is addressed. By coexpression of the two antigens in HuH-7 cells followed by specific antibody precipitation, it was found that HDAgs of different origins interacted without genotypic discrimination. Moreover, in the presence of hepatitis B virus surface antigen, HDAg-S was incorporated into virion-like particles through interaction with HDAg-L without genotype restriction. As to the differences in replication activation of genotype I HDV RNA, all HDAg-S clones tested had some trans-activation activity, and this activity varied greatly among isolates. As to the support of HDV genotype II replication, only clones of HDAg-S from genotype II showed trans-activation activity, and this activity also varied among isolates. In conclusion, genotype has no effect on HDAg interaction and genotype per se only partly predicts how much the HDAg-S of an HDV isolate affects the replication of a second HDV isolate.

Varied assembly and RNA editing efficiencies between genotypes I and II hepatitis D virus and their implications.

The mechanisms that link genotypes of hepatitis D virus (HDV) with clinical outcomes have not yet been elucidated. Genotypic variations are unevenly distributed along the sequences of hepatitis delta antigens (HDAgs). Of these variations, the packaging signal at the C-terminus has a divergence of 74% between genotypes I and II. In this report, we address the issue of whether these high variations between genotypes affect assembly efficiency of HDV particles and editing efficiency of RNA. Viral package systems of transfection with expression plasmids of hepatitis B surface antigen and HDAgs or whole genomes of HDV consistently indicate that the package efficiency of genotype I HDV is higher than that of genotype II. Segment swapping of large-form HDAg indicates that the C-terminal 19-residue region plays a key role for the varied assembly efficiencies. Also, the editing efficiency of genotype I HDV is higher than that of genotype II. The nucleotide and structural changes surrounding the editing site may explain why genotype II HDV has a low RNA editing efficiency. The findings of in vitro assembly systems were further supported by the observations that patients infected with genotype II had significantly lower alanine transaminase (ALT) levels, more favorable outcomes (P <.05), and a trend to have lower serum HDV RNA levels as compared with those infected with genotype I HDV (P =.094). In conclusion, genotype II HDV secretes fewer viral particles than genotype I HDV does, which in turn may reduce the extent of infection of hepatocytes and result in less severe hepatic inflammation.

Spread of hepatitis E virus among different-aged pigs: two-year survey in Taiwan.

Swine are reservoirs of hepatitis E virus (HEV). In this study, a 2-year survey of HEV in feces and sera of swine was conducted to determine if: 1) HEV has circulated among pigs for some time in Taiwan; 2) the spread of HEV among different-aged pigs; and 3) there exists HEV strains possibly imported through trading. From 1998-2000, 521 serum samples and 54 fecal specimens from pigs were examined by reverse transcription polymerase chain reaction. None of the 11 pigs in suckling stage (< 2 months) were serum HEV RNA positive. The highest viremia rate (4.5%) was in pigs of 2 months age, followed by 1.2% and 1.8% in pigs of growing (3-4 months) and finishing stages (5-6 months), and none in pigs older than 6 months. Viremia showed little variation in different years and areas. None of the 20 fecal samples from pigs in suckling stage were HEV RNA positive, whereas 9% of the 34 samples from pigs in growing or finishing stages were positive. Most swine HEV isolates in Taiwan clustered within the genotype 4, whereas the three HEV isolates cloned from pigs imported recently from the U.S. belonged to the genotype 3 HEV in the U.S. The results suggest that HEV may infect pigs at an early growing stage and spread unnoticed among pigs and possibly across countries through trading.