I-Jane Sheen

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 <or= 1cm (3.333, 1.487-7.470) were correlated with early recurrence (within two years of operation) of HCC. In addition, multivariate analysis determined that Ishak hepatic inflammatory activity >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.

Clinical and epidemiological implications of swine hepatitis E virus infection

In nonendemic areas, most patients with acute hepatitis E were infected through traveling to endemic areas. However, some patients did not have a history of foreign travel before infection. Furthermore, high seroprevalence rates of antibody to hepatitis E virus (anti‐HEV) were found in the general adult population in some countries without any recorded outbreak of hepatitis E. The significance of anti‐HEV assay in these subjects remains obscure. To study if swine might be a source of HEV infection, HEV was tested in sera of 235 pigs in Taiwan, and from 5 patients with acute HEV infection who either denied or did not provide any foreign travel history. Three (1.3%) pigs had detectable swine HEV RNA. The swine and human HEV strains from Taiwan formed a monophyletic group, distinct from three previously reported groups: the United States human and swine HEV strains, the Mexico strain, and the largest group composed of the Asian and the African strains. The identity of nucleotide sequences was 84–95% between swine and human HEV strains in Taiwan, and 72–79% between Taiwan strains and those from different areas. The predicted amino acid sequence of a Taiwan swine HEV strain within the peptide 3‐2 used in commercial anti‐HEV assay showed a high identity (91–94%) with those of other human and swine HEV strains. Swine may be a reservoir of HEV and subclinical swine HEV infection may occur. Cross‐reactivity of current anti‐HEV assay may account for the high prevalence rate of anti‐HEV in the general population in nonendemic areas. J. Med. Virol. 60:166–171, 2000.

Characterization and phylogenetic analysis of a novel hepatitis D virus strain discovered by restriction fragment length polymorphism analysis.

The hepatitis D virus (HDV) genotypes in 46 HDV-infected patients and 12 prostitutes were screened with Xhol restriction fragment length polymorphism (RFLP) analysis of reverse transcription PCR products of viral genomes and verified by phylogenetic analysis. The amplificates of three (6.5%) patients and two (17%) prostitutes showed a novel RFLP pattern different from those of the three known genotypes. Complete HDV genomic sequence identities between isolates with a novel RFLP and the HDV genotypes I, II and III were 72.3, 77.2 and 63.0%, respectively. Importantly, divergence was mostly seen in various regions related to replication or packaging. The novel isolates formed a monophyletic group (P < 0.05) and were most closely related to genotype II.

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.

Hepatitis B Surface Antigen Levels and Sequences of Natural Hepatitis B Virus Variants Influence the Assembly and Secretion of Hepatitis D Virus

ABSTRACT 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.

Localization of isoprenylated antigen of hepatitis delta virus by anti-farnesyl antibodies.

Hepatitis delta virus (HDV) is a subviral pathogen that requires pre-existing or concurrent infection with hepatitis B virus (HBV). HDV expresses two forms of a single protein, the delta antigen (HDAg), which are identical except for an additional 19 residues at the C terminus of the large form. Within this C-terminal extension a cysteine residue is isoprenylated; this isoprenylation is critical for interaction with HBV envelope proteins to enable virus assembly and release into the medium. Therefore, large HDAg must be recruited to an extracellular compartment. However, immuno-staining with HDAg-specific antibodies has localized the large antigen mainly to the nucleus and supports the notion that large HDAg suppresses virus replication in the nucleus. Since isoprenylation would increase the hydrophobicity of the protein and may favour transport towards specific membranes, the question remains whether the large HDAg detected in the nucleus carries an isoprenyl group. To address this issue, antibodies against the farnesyl modification were generated to allow direct visualization of the antigen by immunofluorescence microscopy. The anti-farnesyl antibodies specifically stained large HDAg expressed in Huh-7 cells, and the signal was largely restricted to the nucleus; the staining pattern could be superimposed on those of cells stained for large HDAg. The large HDAg translocated into the nucleus was therefore isoprenylated. In addition, antibodies specific for the farnesyl modification should be applicable to the study of other similarly isoprenylated proteins.

Mixed genotypes infection with hepatitis D virus

Heterogeneity of hepatitis C viral (HCV) genomes results in escape from immune clearance. Super‐infection or mixed infection of different genotypes of HCV are seen commonly in humans. Hepatitis D virus (HDV) is classified into 3 genotypes. This study was planned to investigate if mixed genotypes infection of HDV occurs in humans. HDV genotyping based on restriction fragment length polymorphism (RFLP) was used to screen 60–99 HDV clones from each case of 7 prostitutes and 11 patients. Mixed infections were diagnosed by the finding of two or more different RFLP patterns in a case and were confirmed by sequencing. Five prostitutes had mixed infections of genotypes IIa and IIb HDV, while only 2 patients had mixed infections of genotypes I and II HDV (P < 0.05). The heterogeneity in nucleotide sequence was generally below 2% among HDV quasi‐species from the same subject, while the heterogeneity was 27.7% between genotypes I and II HDV, and 22.8% between genotypes IIa and IIb HDV from a subject with mixed infection. Multiple HDV clones from the spouses of the 2 index cases were also analyzed. One spouse had mixed infection and the other did not, corresponding to the index cases. In cases with mixed genotypes infections, the prevalence of the minor strain was less than 10% of the total colony population analyzed. J. Med. Virol. 57:64–67, 1999.

Positive Selection of Hepatitis Delta Antigen in Chronic Hepatitis D Patients

ABSTRACT 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.

Evidence of transmission of hepatitis B virus to spouses from sequence analysis of the viral genome

Heterosexual contact is one of the common routes of transmission for hepatitis B virus (HBV) among adults in Taiwan, but only a few studies have provided direct evidence at the level of the HBV genome of infected couples with acute non‐fulminant hepatitis to document a common source. By cloning and sequencing polymerase chain reaction‐amplified HBV‐DNA, we analysed the sequences of the conserved region of the surface gene (nucleotide (nt) 305–513, representing 6.5% of the viral genome) of HBV in five HBV‐infected index patients, their spouses and four randomly selected HBV carriers as controls. Risk factors associated with acute HBV infection in index cases were sexual contact with their spouses within 3 months before the onset of hepatitis. For all five couples, the HBV‐infected index patient and the spouse shared a 100% sequence homology of HBV‐DNA. In contrast, there was significantly more variation (mean heterogeneity 6.1%, range 1–13.9%) in the amplified region between the five couples and between each couple and the controls (P < 0.001). This study demonstrated that sequence analyses can correlate well with epidemiological findings and confirm the value of the molecular approach for linked infections of HBV through heterosexual contact between spouses. Susceptible adults should receive vaccination.

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.