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HCV-rna positivity in peripheral blood mononuclear cells of patients with chronic HCV-infection: does it really mean viral replication?

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World Journal of Gastroenterology 2001年第2期7卷 228-234页

作者： Volker Meier Sabine Mihm Perdita Wietzke-Braun Guliano Ramadori Georg-August-Universitat gottingen Zentrum innere medizinAbteilung Für Gastroenterologie Und EndokrinologieRobert-Koch-Straβe 4037075 GottingenGermany

AIM: To analyze the association of HCV-rna with peripheral blood mononuclear cells (PBMC) and to answer the question whether HCV-rna positivity in PBMC is due to viral replication. METHODS: HCV-rna was monitored in serum and PBMC preparations from 15 patients with chronic HCV infection before, during and after an IFN-alpha therapy using a nested RT/PCR technique. In a second approach, PBMC from healthy donors were incubated in HCV positive plasma. RESULTS: In the IFN-alpha responding patients,HCV-rna disappeared first from total rna preparations of PBMC and then from serum. In contrast, in relapsing patients, HCV-rna reappeared first in serum and then in PBMC. A quantitative analysis of the HCV-rna concentration in serum was performed before and after transition from detectable to non detectable HCV-rna in PBMC-rna and vice versa. When HCV-rna was detectable in PBMC preparations, the HCV concentration in serum was significantly higher than the serum HCV-rna concentration when HCV-rna in PBMC was not detectable. Furthermore, at no time during the observation period was HCV specific rna observed in PBMC, if HCV-rna in serum was under the detection limit. Incubation of PBMC from healthy donors with several dilutions of HCV positive plasma for two hours showed a concentration dependent PCR positivity for HCV-rna in reisolated PBMC. CONCLUSION: The detectability of HCV-rna in total rna from PBMC seems to depend on the HCV concentration in serum. Contamination or passive adsorption by circulating virus could be the reason for detection of HCV-rna in PBMC preparations of chronically infected patients.

关键词： Adult Aged Antiviral Agents Female Hepacivirus Hepatitis C, Chronic Humans Interferon-alpha Leukocytes, Mononuclear Male Middle Aged rna, viral Research Support, Non-U.S. Gov't Reverse Transcriptase Polymerase Chain Reaction viral Load Virus Replication

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Is laparoscopy an advantage in the diagnosis of cirrhosis in chronic hepatitis C virus infection?

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World Journal of Gastroenterology 2003年第4期9卷 745-750页

作者： Perdita Wietzke-Braun Felix Braun Peter Schott Giuliano Ramadori Department of Internal Medicine Division of Gastroenterology and EndocrinologyUniversity of GoettingenGermany

AIM:To evaluate the potential of laparoscopy in the diagnosis of cirrhosis and outcome of interferon treatment in HCV-infected patients. METHODS:In this retrospective study,diagnostic laparoscopy with laparoscopic liver biopsy was performed in 72 consecutive patients with chronic HCV *** presence or absence of drrhosis was analyzed macroscopically by laparoscopy and microscopically by liver biopsy *** and laboratory data and outcome of interferon-alfa treatment were compared between cirrhotic and noncirrhotic patients. RESULTS:Laparoscopically,cirrhosis was seen in 29.2 % (21/72)and non-cirrhosis in 70.8 %(51/72)of patients. Cirrhotic patients were significantly older with a significant longer duration of HCV infection than noncirrhotic patients. Laboratory parameters(AST,y-GT,y-globulin fraction)were measured significantly higher as well as significantly lower (prothrombin index,platelet count)in cirrhotic patients than in non-cirrhotic ***,cirrhosis was confirmed in 11.1%(8/72)and non cirrhosis in 88.9 %(64/72).Patients with macroscopically confirmed cirrhosis(n=21)showed histologically cirrhosis in 38.2 %(8/21)and histologically non- cirrhosis in 61.9 %(13/21).In contrast,patients with macroscopically non-cirrhosis(n=51)showed histologically non cirrhosis in all cases(51/51).Thirty-nine of 72 patients were treated with interferon-alfa,resulting in 35.9 %(14/39) patients with sustained response and 64.1%(25/39)with non ***-responders showed significantly more macroscopically cirrhosis than sustained *** contrast,there were no significant histological differences between non-responders and sustained responders. CONCLUSION:Diagnostic laparoscopy is more accurate than liver biopsy in recognizing cirrhosis in patients with chronic HCV *** biopsy is the best way to assess inflammatory grade and fibrotic *** invasive marker for staging,prognosis and management,and treatment outcome of chro

关键词： Adult Biopsy Comparative Study Female Genotype Hepacivirus purification Hepatitis C, Chronic Humans Interferon-alpha Laparoscopy Liver Cirrhosis Male Middle Aged rna, viral Reproducibility of Results Retrospective Studies Treatment Outcome

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Hepatitis C virus infection of human hepatoma cell line 7721 in vitro

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World Journal of Gastroenterology 2001年第5期7卷 685-689页

作者： Zhi-Qiang Song~1 Fei Hao~1 Feng Min~2 Qiao-Yu Ma~2 Guo-Dong Liu~2 Department of Dermatology~1Department of Infectious Diseases~2,Southwest Hospital,Third Military Medical University,Chongqing 400038,China Department of Dermatology Southwest Hospital Third Military Medical University Chongqing 400038 China. songzq@***

AIM: To establish a cell culture system with long-term replication of hepatitis C virus in vitro. METHODS: Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various phases during the culturing periods. The presence of HCV rna, the expression of HCV antigens in cells and/or supernatant were examined by RTPCR, in situ hybridization and immunohistochemistry respectively. RESULTS: The intracellular HCV rna was first detected on d2 after infection and then could be intermittently detected in both cells and supernatant over a period of at least three months. The expression of HCV NS3,CP10 antigens could be observed in cells. The fresh cells could be infected by supernatant from cultured infected cells and the transmission of viral genome from HCV-infected 7721 cells to PBMCs was also observed. CONCLUSION: The hepatoma line 7721 is not only susceptible to HCV but also supports its long-term replication in vitro.

关键词： Carcinoma, Hepatocellular Liver Neoplasms Antigens, viral Cell Division Hepacivirus development Hepatitis C Humans In Situ Hybridization In Vitro Phenotype rna, viral Research Support, Non-U.S. Gov't Tumor Cells, Cultured Virus Replication

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Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction

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World Journal of Gastroenterology 2001年第3期7卷 370-375页

作者： Ji Lin Cheng Bao Ling Liu Yi Zhang Wen Bin Tong Zheng Yan Bai Fang Feng Institute of Hepatology,Peoples Hospital,Medical Center of Beijing University,Beijing 10(X)44,China Institute of Hepatology People's Hospital Medical Center of Beijing University of Beijing China.

AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV rna strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. RESULTS: HCV rna positive strands were consistently detected the cultured cells for one year. The negative-strand rna in LCL cells and the positive-strand rna in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight rnase digestion but not DNase digestion. CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.

关键词： B-Lymphocytes Cells, Cultured Female Hepacivirus development purification Herpesvirus 4, Human Humans Immunohistochemistry In Vitro Polymerase Chain Reaction rna, viral Research Support, Non-U.S. Gov't Reverse Transcriptase Polymerase Chain Reaction Transformation, Genetic viral Core Proteins viral Nonstructural Proteins Virus Replication

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SEN virus does not affect treatment response in hepatitis C virus coinfected patients but SEN virus response depends on SEN virus DNA concentration

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World Journal of Gastroenterology 2004年第13期10卷 1893-1897页

作者： Abdurrahman Sagin Ortwin Adams OliverKirschberg AndreasErhardt TobiasHeintges Dieter Hussinger Klinik fr Gastroenterologie Hepatologie und InfektiologieUniversittsklinikum DsseldorfMoorenstr 540225 DsseldorfGermany Institut fr Virologie der Heinrich-Heine-Universitt Dsseldorf Moorenstr 540225 DsseldorfGermany

AIM: To clarify the effect of SEN virus (SENV) infection on a combination therapy including interferon alfa (IFN-α) or pegylated-IFN with ribavirin in patients with chronic hepatitis and the effect of a combination therapy on ***: SENV DNA was determined by polymerase chain reaction in serum samples from 95 patients with chronic hepatitis C. Quantitative analysis was done for SENV H ***: Twenty-one (22%) of 95 patients were positive for SENV DNA. There was no difference in clinical and biochemical parameters between patients with HCV infection alone and coinfected patients. The sustained response rate for HCV clearance after combination therapy did not differ between patients with SENV (52%) and without SENV(50%, n.s.). SENV DNA was undetectable in 76% of the initially SENV positive patients at the end of follow-up. SENV H response to combination therapy was significantly correlated with SENV DNA level (P=-0.05).CONCLUSION: SENV infection had no influence on the HCV sustained response rate to the combination *** rate of SENV to the combination therapy depends on SENV DNA level.

关键词： Adult Antiviral Agents DNA Virus Infections DNA Viruses purification DNA, viral Drug Therapy, Combination Female Hepacivirus Hepatitis C, Chronic Humans Interferon Alfa-2a Interferon-alpha Male Middle Aged Polyethylene Glycols Prevalence rna, viral Ribavirin

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Serum concentration of sFas and sFasL in healthy HBsAg carriers,chronic viral hepatitis B and C patients

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World Journal of Gastroenterology 2004年第24期10卷 3650-3653页

作者： Tadeusz Wojciech Lapinski Oksana Kowalczuk Danuta Prokopowicz Lech Chyczewski Department of Infectious Diseases Medical University of BialystokPoland Institute of Molecular Biology Medical University of BialystokPoland

AIM:To estimate the amount of apoptosis among healthy HBsAg carriers,patients with chronic HBV infection treated wibh lamivudine and patients with chronic HCV infection treated with interferon alpha and *** of apoptosis was evaluated by serum sFas/sFasL concentration measurement. Moreover dependence between apoptosis and HBV-DNA or HCV-rna levels was studied. METHODS:Eighty-six persons were included into study:34 healthy HBsAg carders,33 patients with chronic HBV infecl^on and 19 patients with chronic HCV *** levels of sFas/sFasL were measured by ELISA ***-DNA and HCV-rna were measured by RT-PCR *** of sFas/sFasL were determined before and 2 and 12 wk after therapy in patients with chronic hepatitis B and C infection. HBV-DNA or HCV-rna was detected before treatment and 6 mo after treatment. RESULTS:Twenty-four (71%) healthy HBsAg carders showed HBV-DNA over 10~5/mL,which was comparable to the patients with chronic hepatitis *** from HBV-DNA levels, the concentration of sFas among healthy HBsAg carders was comparable to healthy *** patients with chronic hepatitis B and C,the concentration of sFas was significantly higher in comparison to healthy HBsAg carriers and healthy *** chronic hepatitis B patients the concentration of sFas was decreased during lamivudine *** chronic hepatitis C patients the concentration of sFas was increased during IFN alpha and ribavirin treatment,sFasL was not detected in control *** sFasL occurred more frequently in chronic hepatitis C patients in comparison to chronic hepatitis B patients. CONCLUSION:There are no correlations between apoptosis and HBV-DNA *** ther is an association between apoptosis and activity of inflammation in patients with chronic HBV *** can be increased in patients with chronic hepatitis C by effective treatment which may be a result of apoptosis stimulation by IFN-α.

关键词： Adolescent Adult Aged Antigens, CD95 Apoptosis Biological Markers Carrier State DNA, viral Female Hepatitis B Surface Antigens Hepatitis B, Chronic Hepatitis C, Chronic Humans Lamivudine Male Membrane Glycoproteins Middle Aged rna, viral Reverse Transcriptase Inhibitors Solubility

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Assessment of correlation between serum titers of hepatitis c virus and severity of liver disease

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World Journal of Gastroenterology 2004年第16期10卷 2409-2411页

作者： BhupinderS.Anand MariaVelez Department of Medicine V.A.Medical Center and Baylor College of MedicineHoustonTexas 77030USA

AIM:The significance of hepatitis C virus (HCV) serum titers has been examined in several clinical situations. There is much evidence that patients with a lower viral load have better response rates to anti-viral therapy compared to those with higher ***,a direct association has been observed between serum titers of HCV and transmission rates of the *** aim of the present study was to determine if there was any correlation between HCV viral load and the severity of liver disease. METHODS:Fifty patients with HCV infection were included in the *** comprised of 34 subjects with a history of alcohol use and 16 *** serum HCV rna assay was carried out using the branched DNA (bDNA) *** regression analysis was performed between serum viral titers and liver *** addition,for the purpose of comparison,the subjects were divided into two groups:those with low viral liters (≤50 genome mEq/mL) and high titers (>50 mEq/mL). RESULTS:All subjects were men,with a mean±SD age of 47±7.8 *** mean HCV rna level in the blood was 76.3×10~5±109.1 genome equivalents/*** was no correlation between HCV rna levels and age of the patients (r=0.181),and the history or amount (g/d) of alcohol consumption (r=0.07).Furthermore,no correlation was observed between serum HCV rna levels and the severity of liver disease as judged by the values of serum albumin (r=0.175),bilirubin (r=0.217),ALT (r=0.06) and AST (r=0.004) ***,no significant difference was observed between patients with low viral titers and high liters with respect to any of the parameters. CONCLUSION:Our results indicate that the severity of liver disease is independent of serum levels of hepatitis C *** findings are important since they have a direct impact on the current debate regarding the role of direct cytopathic effect of hepatitis C virus versus immune-mediated injury in the pathogenesis of HCV-related liver damage.

关键词： Adult Alanine Transaminase Alcohol Drinking Aspartate Aminotransferases Diagnosis, Differential Genome, viral Hepacivirus purification Hepatitis C Humans Liver Diseases Liver Function Tests Middle Aged rna, viral Regression Analysis Severity of Illness Index viral Load

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High frequencies of HGV and TTV infections in blood donors in Hangzhou

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World Journal of Gastroenterology 2001年第5期7卷 637-641页

作者： Jie Yan~1 Li-Li Chen~2 Yi-Hui Luo~1 Ya-Fei Mao~1 Meng He~1 1 Department of Pathogenic Biology,Medical School of Zhejiang University,Hangzhou 310006,Zhejiang Province,China2 Department of Stomatology,The Second Affiliated Hospital,Medical School of Zhejiang University,Hangzhou 310009,Zhejiang Province,China Department of Pathogenic Biology Medical School of Zhejiang University 353 Yan An Road Hangzhou 310006 Zhejiang ProvinceChina. yanchen@***

AIM: To determine the frequencies of HGV and TTV infections in blood donors in Hangzhou. METHODS: RT-nested PCR for HGV rna detection and semi-nested PCR for TTV DNA detection in the sera from 203 blood donors, and nucleotide sequence analysis were performed. RESULTS: Thirty-two (15.8%) and 30 (14.8%) of the 203 serum samples were positive for HGV rna and TTV DNA, respectively. And 5 (2.5%) of the 203 serum samples were detectable for both HGV rna and TTV DNA. Homology of the nucleotide sequences of HGV RT-nested PCR products and TTV semi-nested PCR products from 3 serum samples compared with the reported HGV and TTV sequences was 89.36%, 87.94%, 88.65% and 63.51%, 65.77% and 67.12%, respectively. CONCLUSION: The infection rates of HGV and/or TTV in blood donors are relatively high, and to establish HGV and TTV examinations to screen blood donors is needed for transfusion security. The genomic heterogeneity of TTV or HGV is present in the isolates from different areas.

关键词： Base Sequence Blood Donors numerical data Blood Transfusion China DNA, viral GB virus C purification Hepatitis, viral, Human Humans Molecular Sequence Data Polymerase Chain Reaction rna, viral Sequence Analysis, DNA Torque teno virus

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Effect of in vitro interferon-beta administration on hepatitis C virus in peripheral blood mononuclear cells as a predictive marker of clinical response to interferon treatment for chronic hepatitis C

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World Journal of Gastroenterology 2004年第5期10卷 733-736页

作者： Kaori Mochizuki Tatehiro Kagawa Shinji Takashimizu Kazuya Kawazoe Sei-Ichiro Kojima Naruhiko Nagata Atsushi Nakano Yasuhiro Nishizaki Koichi Shiraishi Masaru Itakura Norihito Watanabe Tetsuya Mine Shohei Matsuzaki Department of Internal Medicine Division of GastroenterologyTokai University School of MedicineBohseidaiIsehara 259-1193Japan

AIM:To test whether in vitro incubation of peripheral blood mononuclear cells (PBMC) with interferon (IFN) could efficiently decrease hepatitis C virus-rna (HCV-rna) amount and to analyze whether this effect was associated with clinical response to ***:Twenty-seven patients with histologically proven chronic hepatitis C were given intravenous administration of 6 million units (MU) IFN-β daily for 6 weeks followed by three times weekly for 20 weeks. PBMC collected before IFN therapy were incubated with IFN-β and HCV-rna in PMBC was semi-quantitatively ***: Twenty-five patients completed IFN *** patients (32%) had sustained loss of serum HCV-rna with normal serum ALT levels after IFN therapy (complete responders).HCV-rna in PBMC was detected in all patients,whereas it was not detected in PBMC from healthy *** vitro administration of IFN-β decreased the amount of HCV-rna in PMBC in 18 patients (72%). Eight of these patients obtained complete response. On the other hand,none of the patients whose HCV-rna in PBMC did not decrease by IFN-β was complete responders. Multiple logistic regression analysis revealed that the decrease of HCV-rna amount in PBMC by IFN-β was the only independent predictor for complete response (P<0.05).CONCLUSION:The effect of in vitro IFN-β on HCV in PBMC reflects clinical response and would be taken into account as a predictive marker of IFN therapy for chronic hepatitis C.

关键词： Adult Antiviral Agents dosage Drug Resistance, viral Female Hepacivirus Hepatitis C, Chronic Humans In Vitro Interferon-beta Leukocytes, Mononuclear Male Middle Aged Predictive Value of Tests rna, viral

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Partial nucleotide sequencing of hepatitis E viruses detected in sera of patients with hepatitis E from 14 cities in China

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Chinese Medical Journal 2002年第7期115卷 1058-1063,152-153页

作者：李奎庄辉朱万孚北京大学医学部微生物学系北京100083

OBJECTIVE: To investigate the genotypes of hepatitis E viruses (HEV) detected in sera of patients from different regions of China. METHODS: The partial genome (nt6461-6860, nt5994-6294) of open reading frame 2 (ORF2) of 45 HEV strains detected from 14 cities of China was amplified and sequenced using polymerase chain reaction (PCR) and direct sequencing. RESULTS: Forty-one of 45 strains (91%) share the same genotype with HEV Burma strain (B), with nucleotide identities higher than 98% with the representative HEV Chinese strain. Only 4 HEV strains are significantly divergent from the 3 prototype strains of HEV, with nucleotide identities of 77%-80% with HEV Burmese/Chinese strain, 74%-76% with Mexican strain and 74%-77% with the newly discovered HEV US/swine strain, respectively. Phylogenetic analysis suggests that these 4 strains may represent 2 different subtypes that belong to a novel genotype of HEV, which is significantly divergent from the prototype Mexico, Burmese and US/swine strains. CONCLUSION: Among patients with hepatitis E in China, most are infected by the Chinese prototype HEV, and only a small part by the new genotype HEV.

关键词： Base Sequence Genotype Hepatitis E Hepatitis E virus Humans Open Reading Frames Phylogeny rna, viral Research Support, Non-U.S. Gov't

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