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Identification and characterization of HBSAG variants in chronic hepatitis B virus carriers who lost HBSAG during follow-up
Category 5: Viral hepatitis: basic aspects there was cross-linked relationship the associated mutation of 1272/1287 to 1126/l 134 (p
I
382
IDENTIFICATION VARIANTS
AND CHARACTERIZATION
IN CHRONIC
LOST HBSAG DURING
OF HBSAG
HEPATITIS B VIRUS CARRIERS
WHO
FOLLOW-UP
K.H. Kim’, C.Y. Chon2,K.H.Han2,H.Y. Chang’, J.Y. Park2, J.K. Kim2, Y.N. Park3, Y.M. Moon2. ’Yonsei Medical Research Center and Brain Korea 21
Projectfor Medical
of Gastroenterology;
‘Dept.
Science;
2Dept. of Internal Medicine,
Seoul, Korea
has been known that mutations in ‘a’-determinant are related of HBsAg detection. The aim of this study is to identify and the molecular basis of HBsAg and the HBV subtypes from patients. Materials and Methods: A total of 430 chronic HBV carriers were prospectively studied. Liver specimens were obtained after HBsAg-loss in 18 patients. Surface gene was amplified with PCR from sera and liver specimens. The PCR products were cloned to the mammalian expression vector and their sequence mutations were analyzed. After transfection in HepG2 cells, the affinity of cloned HBsAg to antibody was tested. Results: During the follow-up, 49 patients (11.4%) lost serum HBsAg. Among them, 18 cases were analyzed for the HBsAg characterization study. Thirty-six percent had neither anti-HBs nor HBsAg. Nevertheless, all of them showed positive for HBV DNA by PCR both in liver tissue and serum. Mutations were found in the ‘a’-determinant region. The HBsAg mutations from the serum samples before and after the HBsAg-loss were determined with 3 patients. The expression of cloned HBsAg with ‘a’-determinant mutations showed lower affinity to the anti-HBs than wild type. Among 18 patients, interestingly, 14 patients (78%) were ayw subtype. Conclusions: Our results demonstrate that the persistent presence of HBV after loss of HBsAg might be closely related with emerging mutations in HBsAg especially in the ‘a’-determinant region and with the specific subtype, ayw, in Korea.
383
INJECTION
OF HBV-DNA
T-CELL RESPONSE
RESULTS
IN REPLICATION
AND
IN MICE
C. Klein’,
T. Bock2, T. Wuestefeld’, H. Wedemeier’, H.P. Diennes3, S. Locarnini4, M.l? Manns’, C. Trautwein’. ‘Medical School Hannoves Gastroenterology
Hepatology
And Endocrinology,
2 University Tuebingen,
Institute Of Pathology,
’ Univerity Of Kologne,
Kologne,
Melbourne,
Germany;
I 384
QUANTITATIVE
DETECTION
AND REPLICATIVE
HEPATITIS B VIRUS CCCDNA
It Purpose: to the failure characterize HBsAg-loss
I
In the liver of the animals viral-RNA expression was found. Maximum HBV-RNA-expression was found 3 days after injection. Replicative HBVintermediates were detected in viral particles from digested liver. Polymerase activity was monitored by endogenous polymerase assay. Additional progeny-DNA was detected by Southern Blot analysis of viral partcles in the liver and by PCR in the serum. Highest polymerase activity was found 3 days after injection. We found transduction rates of about 5.10% HBsAg-positive hepatocytes 2 days after injection. Injection of HBV-DNA resulted in a significant T-cell response showed by IFNy elispot assays which was associated with an increase of transaminases. With the established new protocol we are enable to study the replication pattern of HBV mutants and the impact of new anti-viral strategies on HBV replication
Inst.
of Pathology, Inst. of Gastroenterology,
Yonsei Univ. College of Medicine,
113
Hannoves
Tuebingen,
ACTIVITY
IN THE LIVER IN CHRONIC
OF HBV
INFECTION
A. Laras’,
E. Dimou2, S.J. Hadziyannis2.
Medicine,
Hepatology
Medicine,
18, Papadiamantopoulou
Research
‘Second
Department
Of
Lab, Athens University School Of Sk, Athens, Greece;
Henry Dunant Hospital, 107, Mesogeion
2Liver Unit,
Sk:, Athens, Greece
Background: Measurement of pregenomic RNA (pgRNA) levels in conjunction with cccDNA in the liver may provide a more precise way for evaluating HBV replication than cccDNA levels alone. Methods: HBV cccDNA, total HBV DNA and pgRNA were quantified in liver biopsies from 20 [HBeAg (+) 8, HBeAg (-) 121 patients (pts) with chronic HBV infection with a new validated quantitative real-time PCR and RT-PCR technique. Results were normalized to cellular beta-globin levels. HBV DNA cccDNA replicative activity was expressed as molecules of pgRNA per cccDNA. Results: HBV cccDNA was detected in 17/20 samples, while pgRNA in all. The median copy number of cccDNA per cell was 0.4 (0.003-11.6), while the median number of pgRNA transcripts per cell 1.8 (0.008-1046). The median values for cccDNA and pgRNA levels in HBeAg (-) pts were respectively 20.fold (93 x lo3 vs. 2100 x 103) and lOOO-fold (350 x lo3 vs. 470000 x 103) lower than in HBeAg (+). The median number of pgRNA copies per cccDNA was significantly higher in the HBeAg (+) vs. the HBeAg (-) pts: 116 (23-788) vs. 8 (0.5-24). Conclusions: 1 Measurement of pgRNA in the liver provides more accurate and lo-100 fold more sensitive information on viral replication compared to the cccDNA levels. 2 Patients with precore and/or core promoter mutants exhibit significantly lower transcriptional and replicative activity than pts with wild-type (wt) HBV. 3 Combined measurement of cccDNA and pgRNA provides important information on residual HBV and the efficacy of antiviral therapy both in wt and precore mutant HBV infection.
Germany;
Germany;
4 University Of Melbourne,
I 385
Tail-vein-injection of plasmid-DNA in mice enables exogenous gene expression in hepatocytes. At present an mouse model for studying hepatitis B virus gene expression, replication and the native T-cell response is missing. We established a mouse-model by tail-vein-injection of HBV-DNA. Replication competent plasmid-DNA containing the l.SxHBV-genome was injected in mice. Repeated injections with different doses/volumes were performed to raise the transduction and expression efficacy. At different time points after injection HBV-markers in the serum and the liver of the animals were analysed. At different time points after injection of pHBV1.5.plasmid in mice, serum and mouse liver were analysed for HBV markers. HBsAg, HBeAg expression increased during the first 3 days after injection to maximal levels. During 14 days HBsAg+ and HBeAg+ titers were found.
THE NS3 PROTEASE
GENE OF HCV IS HIGHLY CONSERVED
WITHIN THE PUTATIVE CATALYTIC SITE REGION
Australia
S. Lodrini’, S. Bagaglio’, F. Canducci2, M.S. De Mitri3, A. Lazzarin’, M. Clementi2, G. Morsica’. ‘Division Of Infectious Diseases, San Raffaele Scient$c Raffaele’,
Internal Medicine, Bologna,
Institute, Milan, Italy; 2 University
‘Vita-Salute San
San Raffaele Scientz$c Institute, Milan, Italy; ‘Department Cardioangiology,
Hepatology,
Of
University Of Bologna,
Italy
Background and Aim: Few data are available on the genetic diversity of the NS3 protease gene that presents a potential target for antiviral therapy. We developed a nested PCR to obtain NS3 protease sequences from patients infected with different HCV genotypes. Methods: The NS3 region of HCV was directly sequenced and alignment of nucleotide and amino acid sequences was performed by ClustalX. Phylogenetic tree was constructed by Phylip package.
I
382
IDENTIFICATION VARIANTS
AND CHARACTERIZATION
IN CHRONIC
LOST HBSAG DURING
OF HBSAG
HEPATITIS B VIRUS CARRIERS
WHO
FOLLOW-UP
K.H. Kim’, C.Y. Chon2,K.H.Han2,H.Y. Chang’, J.Y. Park2, J.K. Kim2, Y.N. Park3, Y.M. Moon2. ’Yonsei Medical Research Center and Brain Korea 21
Projectfor Medical
of Gastroenterology;
‘Dept.
Science;
2Dept. of Internal Medicine,
Seoul, Korea
has been known that mutations in ‘a’-determinant are related of HBsAg detection. The aim of this study is to identify and the molecular basis of HBsAg and the HBV subtypes from patients. Materials and Methods: A total of 430 chronic HBV carriers were prospectively studied. Liver specimens were obtained after HBsAg-loss in 18 patients. Surface gene was amplified with PCR from sera and liver specimens. The PCR products were cloned to the mammalian expression vector and their sequence mutations were analyzed. After transfection in HepG2 cells, the affinity of cloned HBsAg to antibody was tested. Results: During the follow-up, 49 patients (11.4%) lost serum HBsAg. Among them, 18 cases were analyzed for the HBsAg characterization study. Thirty-six percent had neither anti-HBs nor HBsAg. Nevertheless, all of them showed positive for HBV DNA by PCR both in liver tissue and serum. Mutations were found in the ‘a’-determinant region. The HBsAg mutations from the serum samples before and after the HBsAg-loss were determined with 3 patients. The expression of cloned HBsAg with ‘a’-determinant mutations showed lower affinity to the anti-HBs than wild type. Among 18 patients, interestingly, 14 patients (78%) were ayw subtype. Conclusions: Our results demonstrate that the persistent presence of HBV after loss of HBsAg might be closely related with emerging mutations in HBsAg especially in the ‘a’-determinant region and with the specific subtype, ayw, in Korea.
383
INJECTION
OF HBV-DNA
T-CELL RESPONSE
RESULTS
IN REPLICATION
AND
IN MICE
C. Klein’,
T. Bock2, T. Wuestefeld’, H. Wedemeier’, H.P. Diennes3, S. Locarnini4, M.l? Manns’, C. Trautwein’. ‘Medical School Hannoves Gastroenterology
Hepatology
And Endocrinology,
2 University Tuebingen,
Institute Of Pathology,
’ Univerity Of Kologne,
Kologne,
Melbourne,
Germany;
I 384
QUANTITATIVE
DETECTION
AND REPLICATIVE
HEPATITIS B VIRUS CCCDNA
It Purpose: to the failure characterize HBsAg-loss
I
In the liver of the animals viral-RNA expression was found. Maximum HBV-RNA-expression was found 3 days after injection. Replicative HBVintermediates were detected in viral particles from digested liver. Polymerase activity was monitored by endogenous polymerase assay. Additional progeny-DNA was detected by Southern Blot analysis of viral partcles in the liver and by PCR in the serum. Highest polymerase activity was found 3 days after injection. We found transduction rates of about 5.10% HBsAg-positive hepatocytes 2 days after injection. Injection of HBV-DNA resulted in a significant T-cell response showed by IFNy elispot assays which was associated with an increase of transaminases. With the established new protocol we are enable to study the replication pattern of HBV mutants and the impact of new anti-viral strategies on HBV replication
Inst.
of Pathology, Inst. of Gastroenterology,
Yonsei Univ. College of Medicine,
113
Hannoves
Tuebingen,
ACTIVITY
IN THE LIVER IN CHRONIC
OF HBV
INFECTION
A. Laras’,
E. Dimou2, S.J. Hadziyannis2.
Medicine,
Hepatology
Medicine,
18, Papadiamantopoulou
Research
‘Second
Department
Of
Lab, Athens University School Of Sk, Athens, Greece;
Henry Dunant Hospital, 107, Mesogeion
2Liver Unit,
Sk:, Athens, Greece
Background: Measurement of pregenomic RNA (pgRNA) levels in conjunction with cccDNA in the liver may provide a more precise way for evaluating HBV replication than cccDNA levels alone. Methods: HBV cccDNA, total HBV DNA and pgRNA were quantified in liver biopsies from 20 [HBeAg (+) 8, HBeAg (-) 121 patients (pts) with chronic HBV infection with a new validated quantitative real-time PCR and RT-PCR technique. Results were normalized to cellular beta-globin levels. HBV DNA cccDNA replicative activity was expressed as molecules of pgRNA per cccDNA. Results: HBV cccDNA was detected in 17/20 samples, while pgRNA in all. The median copy number of cccDNA per cell was 0.4 (0.003-11.6), while the median number of pgRNA transcripts per cell 1.8 (0.008-1046). The median values for cccDNA and pgRNA levels in HBeAg (-) pts were respectively 20.fold (93 x lo3 vs. 2100 x 103) and lOOO-fold (350 x lo3 vs. 470000 x 103) lower than in HBeAg (+). The median number of pgRNA copies per cccDNA was significantly higher in the HBeAg (+) vs. the HBeAg (-) pts: 116 (23-788) vs. 8 (0.5-24). Conclusions: 1 Measurement of pgRNA in the liver provides more accurate and lo-100 fold more sensitive information on viral replication compared to the cccDNA levels. 2 Patients with precore and/or core promoter mutants exhibit significantly lower transcriptional and replicative activity than pts with wild-type (wt) HBV. 3 Combined measurement of cccDNA and pgRNA provides important information on residual HBV and the efficacy of antiviral therapy both in wt and precore mutant HBV infection.
Germany;
Germany;
4 University Of Melbourne,
I 385
Tail-vein-injection of plasmid-DNA in mice enables exogenous gene expression in hepatocytes. At present an mouse model for studying hepatitis B virus gene expression, replication and the native T-cell response is missing. We established a mouse-model by tail-vein-injection of HBV-DNA. Replication competent plasmid-DNA containing the l.SxHBV-genome was injected in mice. Repeated injections with different doses/volumes were performed to raise the transduction and expression efficacy. At different time points after injection HBV-markers in the serum and the liver of the animals were analysed. At different time points after injection of pHBV1.5.plasmid in mice, serum and mouse liver were analysed for HBV markers. HBsAg, HBeAg expression increased during the first 3 days after injection to maximal levels. During 14 days HBsAg+ and HBeAg+ titers were found.
THE NS3 PROTEASE
GENE OF HCV IS HIGHLY CONSERVED
WITHIN THE PUTATIVE CATALYTIC SITE REGION
Australia
S. Lodrini’, S. Bagaglio’, F. Canducci2, M.S. De Mitri3, A. Lazzarin’, M. Clementi2, G. Morsica’. ‘Division Of Infectious Diseases, San Raffaele Scient$c Raffaele’,
Internal Medicine, Bologna,
Institute, Milan, Italy; 2 University
‘Vita-Salute San
San Raffaele Scientz$c Institute, Milan, Italy; ‘Department Cardioangiology,
Hepatology,
Of
University Of Bologna,
Italy
Background and Aim: Few data are available on the genetic diversity of the NS3 protease gene that presents a potential target for antiviral therapy. We developed a nested PCR to obtain NS3 protease sequences from patients infected with different HCV genotypes. Methods: The NS3 region of HCV was directly sequenced and alignment of nucleotide and amino acid sequences was performed by ClustalX. Phylogenetic tree was constructed by Phylip package.