1063 Quantitative analysis of hepatitis B virus replicative intermediates in the liver: a comparison of pregenomic RNA vs cccDNA comparison of pregenomic RNA vs cccDNA detection

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AASLD ABSTRACTS

HEPATOLOGY, October 2003

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QUANTITATIVE ANALYSIS OF HEPATITIS B VIRUS REPLICATIVE INTERMEDIATES IN THE LIVER: A COMPARISON OF PREGENOMIC RNA VS CCCDNA DETECTION. Andreas Laras, Athens University School of Medicine,

INVOLVEMENT OF HEPATITIS E VIRUS INFECTION IN NON-ABC ACUTE HEPATITIS IN JAPAN. Koji Yano, Yoko

Athens, Greece;~ vangelini Dimou, Henry Dunant Hospital, Athens, Greece; Ageliki Kostamena, John Koskinas, Athens University School of Medicine, Athens, Greece; Stephanos J Hadziyannis, Henry Dunant Hospital, Athens, Greece Background. Replication of hepatitis B virus (HBV) occurs via a covalently closed circular (ccc) DNAintermediate which is responsible for the production of both pregenomic (pg) RNA and viral mRNA transcripts. HBV cccDNA is thought to be responsible for viral persistence in the natural course of chronic HBV infection and during prolonged antiviral therapy. Production of pgRNA from cccDNA is a key step in the HBV life cycle and multiple pgRNA copies are produced from each cccDNA molecule. We and others have recently developed sensitive quantitative assays for HBV cccDNA detection, however precise assessment of intrahepatic cccDNA concentration can be problematic due to its low copy number. Our development of a quantitative transcript-specific assay which allows the in vivo detection of pregenomic RNA molecules provides an alternative method for the quantification of HBV replicative intermediates in the liver of chronically infected patients. Aims. To compare the performance of the two detection methods, pgRNA vs. cccDNA, in a large number of samples in order to determine the most reliable methodology for evaluating HBV persistence and activity in infected patients. Methods. HBV pgRNA and cccDNA levels were quantified by real-time PCR using the Roche LightCycler. Total RNA and DNA were simultaneously extracted from liver biopsy samples of 40 patients with chronic HBV infection, 10 HBeAg-positive (+) and 30 HBeAgnegative (-). HBV pgRNA levels were determined utilizing a transcript-specific RT-PCR assay which monitors the HBV core promoter activity and allows the differential detection and quantification of precore mRNA and pgRNA transcripts. HBV DNA copy number was determined using PCR primers that specifically detect cccDNA or total HBV DNA (and fluorescent hybridization probes). Results were normalized to cellular beta-globin levels. The nucleotide sequence of the HBV core promoter and precore region was determined for each patient. Results. HBV pgRNA was detected in all samples tested, with median copy number of pgRNA transcripts 1.1 per ceil ranging from 0.001-1046 copies (detection limit 0.0003 copies per cell). HBV cccDNA was detectable in all HBeAg(+) patients but only in 21 (70%) of the 30 HBeAg(-) patients. The median copy number of cccDNA was 0.27 per cell, ranging from 0.003-11.6 copies (detection limit 0.003 copies per cell). The mean values for pgRNA and cccDNA levels in HBeAg(-) patients were respectively 1000-fold (0.31 vs. 470) and 20-fold (0.086 vs. 2.1) lower than in HBeAg(+) patients. However pgRNA levels in all but one sample were consistently higher (4- to 788-fold) than their corresponding cccDNA levels. Conclusions. 1. The two methods for quantitative detection of pgRNA and cccDNA in liver biopsy samples yield consistent results that correlate with viral activity and the clinical phase of the infected patients. 2. Combined measurement of pgRNA and cccDNA levels, although cumbersome, provides valuable information on the replicative activity of intrahepatic HBV cccDNA. 3. Measurement of pgRNA has significantly higher sensitivity particularly in HBeAg(-) patients harboring core promoter and/or precore mutant strains with low or undetectable serum HBV DNA levels. 4. We therefore propose that measuring in vivo pgRNA rather than cccDNA levels provides a more sensitive and precise method for evaluating HBV persistence and replication in chronically infected patients. Disclosures: Evangelini Dimou - No relationships to disclose Stephanos J Hactziyannis - No relationships to disclose John Koskinas - No relationships to disclose Ageliki Kostamena - No relationships to disclose Andreas Laras - No relationships to disclose

Tamada, Hiroshi Yatsuhashi, Manabu Daikoku, National Nagasaki Medical Center, Nagasaki, Japan; Michiaki Koga, National Ureshino Hospital, Ureshino, Japan; Hiromi Ishibashi, Michitami Yano, National Nagasaki Medical Center, Nagasaki, Japan Background/Aims: Although hepatitis E is endemic in many Asian countries, clinical hepatitis E infection has been rarely reported in Japan. Most hepatitis E cases observed in Japan have thus far been regarded as "imported". Recently, an HEV strain of genotype III was isolated from a Japanese patient with acute hepatitis who had never been abroad. Consequent studies revealed that Japan indigenous strain might be circulating in Japan. However, the prevalence and the extent of the genetic diversity of HEV in non-ABC acute hepatitis remain to be elucidated. To reveal the role on hepatitis E virus in non-ABC acute hepatitis, immunological and genetical analyses were conducted on patients with non-ABC acute hepatitis. Methods: Stored sera of a nationwide, prospective survey on acute hepatitis, in which 20 national hospitals participated, were utilized in the study. Diagnosis of non-ABC acute hepatitis was made by ruling out hepatitis A, B, and C, chronic hepatitis, alcoholic liver diseases, and autoimmune liver diseases. Sera drawn on admission were collected and stored at -20 °C until being examined. We tested the sera of consecutive 311 patients with non-ABC acute hepatitis, during 1990-2002. Immunoglobulin (Ig) G and M class antibodies were detected by an enzyme immunoassay (EIA). HEV-RNA was detected by polymerase chain reaction with two i n d e p e n d e n t sets of primers derived from well-conserved genomic areas in open reading frame (ORF) I and 2 of HEV genome. Direct sequencing and phylogenetic analysis were performed based on the partial nucleotides sequence of the ORF 1 and ORF 2 region. Results: Fifty seven (18.3%) were positive for IgG class antibodies and 9 (2.9%) cases were positive for IgM antibodies to HEV by EIA. All IgM positive cases were also positive for IgG. HEV IgM positive rate was more prevalent in male (5.1%) than in female (1.5%) and significantly more frequent in 2000-2002 (7.2%) than in 1990-1999 (1.7%). HEV-RNA was detectable in 8 out of 9 IgMpositive samples. Phylogenetic analysis revealed that six, one, and one HEV isolates were of genotypes IIL IV, and L respectively. The patient with genotype I infection had traveled to Bangladesh. Conclusions: These results indicate that polyphyletic HEV is responsible for development of acute hepatitis in Japan. Despite the low endemicity, attention should be paid for the increasing frequency of hepatitis E in non-ABC acute hepatitis. Disclosures: Manabu Daikoku - No relationships to disclose Hiromi Ishibashi - No relationships to disclose Michiaki Koga - No relationships to disclose Yoko Tamada - No relationships to disclose Koji Yano - No relationships to disclose Michitami Yano - No relationships to disclose Hiroshi Yatsuhashi - No relationships to disclose

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SEN VIRUS INFECTION INFLUENCE THE PATHOLOGICAL FINDINGS IN LIVER, BUT NOT AFFECT THE INCIDENCE OF CARCINOMA. Mild Kaneko, Mitsuhiko Moriyama, Hiroshi

Matsumura, Hitomi Nakamura, Shyu Oshiro, Hiroshi Aoki, Atsuo Shioda, Yasuyuki Arakawa, School of Medicine, Nihon University, Tokyo, Japan Aim; In 2001, Tanaka et al. reported the isolation and identification of a new group of transmissible single-stranded DNA viruses, n a m e d SEN virus (SEN-V), which is related to the TTV family. However, no study has examined whether SEN-V infection influences the histological features of the livers and long-term outcome in patients with chronic hepatitis C. In this investigation, we prepared a primer set for the untranslated region of SEN-V, and detected SEN-V DNA in the sera of patients with type C liver disease using a nested PCR. Then, we compared the histological