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Publications (18)101.93 Total impact

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    ABSTRACT: The addition of GB virus C (GBV-C) E2 protein to cells inhibits HIV replication in vitro, presumably triggered by interactions with a specific cellular receptor. Indirect evidence suggests that CD81 is the GBV-C E2 cellular receptor. We found that E2 binding to cells was not dependent upon human CD81, and that soluble CD81 did not compete with GBV-C E2 for cell binding. GBV-C E2 protein thus does not appear to interact with CD81.
    AIDS 06/2007; 21(8):1045-8. · 6.41 Impact Factor
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    ABSTRACT: Epidemiological studies have revealed an association between GB virus C (GBV-C) long-term viraemia and ameliorated HIV disease progression. We have provided evidence that a single protein of GBV-C, the glycoprotein E2, interferes with early HIV replication steps of both X4- and R5-tropic HIV strains. Preincubation with anti-E2 antibody specifically abrogates the inhibitory effect. Results were confirmed by the in-vitro expression of GBV-C E1/E2 encoding RNA.
    AIDS 04/2007; 21(5):645-7. · 6.41 Impact Factor
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    ABSTRACT: GB virus type C (GBV-C) is a human flavivirus that may cause persistent infection, although most infected individuals clear viremia and develop antibodies to the envelope glycoprotein E2. To study GBV-C E2 antigenicity and cell binding, murine anti-E2 monoclonal antibodies (MAbs) were evaluated to topologically map immunogenic sites on GBV-C E2 and for the ability to detect or block recombinant E2 binding to various cell lines. Five competition groups of MAbs were identified. Groups I and II did not compete with each other. Group III competed with both groups I and II. Group IV did not compete with group I, II, or III. One MAb competed with all of the other MAbs, suggesting that the epitopes bound by these MAbs are intimately related. Individually, none of the MAbs competed extensively with polyclonal human convalescent antibody (PcAb); however, combinations of all five MAb groups completely blocked PcAb binding to E2, suggesting that the epitopes bound by these MAbs form a single, immunodominant antigenic site. Only group I and III MAbs detected purified recombinant E2 bound to cells in binding assays. In contrast, group II MAbs neutralized the binding of E2 to cells. Both PcAb and MAbs were conformation dependent, with the exception of one group II MAb (M6). M6 bound to a five-amino-acid sequence on E2 if the peptide included four C-terminal or eight N-terminal residues, suggesting that the GBV-C E2 protein contains a single immunodominant antigenic site which includes a complex epitope that is involved in specific cellular binding.
    Journal of Virology 01/2007; 80(24):12131-40. · 5.08 Impact Factor
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    ABSTRACT: Background: Detection of either GB Virus C RNA or antibody against the GBV-C envelope glycoprotein E2 have been associated with prolonged HIV survival. E2 antibody usually develops at the time of clearance of GBV-C viremia, and is thought to be neutralizing. The role of the two GBV-C envelope glycoproteins in virus-cell interactions and the antigenic specificity of E2 in human infections are not well characterized. Methods: Recombinant GBV-C E2 proteins were expressed in E.coli: complete E1, complete E2 (without p5.6 fragment), and delta-E2 (lacking C-terminal hydrophobic domain). In addition, purified, CHO cell-expressed E2 protein was studied. E2 binding to MOLT-4 cells was evaluated by flow cytometry, and by 125I-labeled E2 cell binding. HCV E2 protein served as the positive control. E2 antibody was detected by the μPlate HGenv test and by dot-blot immunoassay. 6 monoclonal anti-E2 antibodies (McAbs) and numerous human polyclonal sera were evaluated. One of these antibodies (Mc6) was shown to bind viral particles in serum, and to react with a linear epitope on E2. Results: ELISA screening of human sera identified 22 E2 positive samples. 5 negative control sera were used. None of 5 negative controls bound purified E2, whereas 17 of the 22 ELISA positive sera recognized purified E2 in the dot blot. None of these 17 sera recognized denatured E2. Three of the McAbs recognizing conformational epitopes bound E2 but not denatured E2; whereas Mc6 bound purified E2 with and without denaturation. Mc6 binding to E2 was not blocked by polyclonal E2+ sera, thus this epitope does not appear to be immunogenic in humans. Purified recombinant GBV-C E2 protein did not bind to MOLT-4 cells whereas control HCV E2 protein did. Conclusions: A linear epitope on GBV-C E2 protein elicits antibodies that bind virus particles, yet does not appear to elicit antibodies during natural infection. In contrast to HCV E2 protein, purified recombinant E2 protein does not appear to bind MOLT-4 cells, potentially reflecting improper folding of E2 protein. Further studies using the different E2 preparations and virus particles to dissect GBV-C – cell interactions are underway.
    Infectious Diseases Society of America 2003 Annual Meeting; 10/2003
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    ABSTRACT: The risk and clearance of GB virus type C (GBV-C)/hepatitis G virus (HGV) infection was investigated in a cohort of homosexual men (n=180; median follow-up time, 7 years). The interaction between GBV-C/HGV RNA and antibodies against the E2 region of the virus, and the clinical impact of chronic GBV-C/HGV infection were studied. GBV-C/HGV RNA was detected by RT-PCR, and E2 antibodies were assessed by an immunoassay. At baseline, 63% of the participants had evidence of previous or current GBV-C/HGV infection. The GBV-C/HGV incidence rate was 2 per 100 person-years (95% confidence interval 0. 9-3.8) and was similar to the HIV incidence. The incidence of GBV-C/HGV infection was significantly higher in those reporting unprotected anal intercourse (3.6 per 100 person-years compared to 0 in the group without such sexual contacts). The occurrence of E2 antibodies was strongly associated with GBV-C/HGV RNA clearance. A loss of E2 antibodies was observed at a rate of 1.5 per 100 person-years. It was higher among HIV-infected individuals. Chronic GBV-C/HGV infection was not associated with clinical or biochemical evidence of liver disease.
    Journal of Medical Virology 12/1999; 59(3):303-6. · 2.37 Impact Factor
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    ABSTRACT: The risk and clearance of GB virus type C (GBV-C)/hepatitis G virus (HGV) infection was investigated in a cohort of homosexual men (n=180; median follow-up time, 7 years). The interaction between GBV-C/HGV RNA and antibodies against the E2 region of the virus, and the clinical impact of chronic GBV-C/HGV infection were studied. GBV-C/HGV RNA was detected by RT-PCR, and E2 antibodies were assessed by an immunoassay. At baseline, 63% of the participants had evidence of previous or current GBV-C/HGV infection. The GBV-C/HGV incidence rate was 2 per 100 person-years (95% confidence interval 0.9–3.8) and was similar to the HIV incidence. The incidence of GBV-C/HGV infection was significantly higher in those reporting unprotected anal intercourse (3.6 per 100 person-years compared to 0 in the group without such sexual contacts). The occurrence of E2 antibodies was strongly associated with GBV-C/HGV RNA clearance. A loss of E2 antibodies was observed at a rate of 1.5 per 100 person-years. It was higher among HIV-infected individuals. Chronic GBV-C/HGV infection was not associated with clinical or biochemical evidence of liver disease. J. Med. Virol. 59:303–306, 1999. © 1999 Wiley-Liss, Inc.
    Journal of Medical Virology 09/1999; 59(3):303 - 306. · 2.37 Impact Factor
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    ABSTRACT: The clinical significance and course of acute hepatitis G virus (HGV) infection were studied by measuring HGV RNA and antibody to HGV envelope protein E2 (HGV-E2 antibody). A total of 59 patients with transfusion-associated non-A, non-B hepatitis, who were followed-up for more than 1 year, were selected retrospectively. HGV RNA was measured by reverse transcriptase (RT) and nested polymerase chain reaction (PCR) was performed, using primer sets, in the 5'-non-coding region of the HGV genome. HGV-E2 antibody was measured by enzyme-linked immunosorbent assay (ELISA) using recombinant E2 protein. Of the 59 patients, 51 (86%) were infected with hepatitis C virus (HCV) and 12 (20%) were infected with HGV; 11 of the 12 with HGV infection were also infected with HCV. HGV viraemia was cleared during the follow-up period in seven of the 12 patients with HGV infection. All these seven patients seroconverted for HGV-E2 antibody just before or just after the clearance of HGV viraemia. In contrast, all five patients without clearance of HGV viraemia were negative for HGV-E2 antibody (P = 0.0013). Of seven patients with continuous HGV viraemia at 1 year from the onset of acute hepatitis, four with HCV RNA showed chronic elevation of alanine aminotransferase (ALT) but three without HCV RNA did not. The severity of acute hepatitis was similar between patients with both HGV and HCV infections and in those with HCV infection alone. The majority of patients with HGV infection cleared the virus during long-term follow-up. Appearance of HGV-E2 antibody was associated with the clearance of HGV viraemia. An abnormal ALT level was noted to depend on HCV infection but not on HGV infection in both the acute and chronic phases of transfusion-associated hepatitis.
    Journal of Viral Hepatitis 06/1998; 5(3):153-9. · 3.08 Impact Factor
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    ABSTRACT: In order to elucidate the structure and morphology of hepatitis G virus (HGV), a recently isolated flavivirus, we generated a panel of eight monoclonal antibodies (MAbs) against the putative second envelope protein (E2) following DNA immunization. The MAbs were shown to be specific for four different epitopes on recombinant E2. MAb Mc6 was the only antibody able to detect the linear epitope LTGGFYEPL. In addition, Mc6 was able to immunoprecipitate viral particles in human blood samples as detected by reverse transcription-PCR amplification of HGV RNA. This precipitation could be competed by addition of saturating amounts of the linear peptide or abolished by addition of Nonidet P-40. We conclude that, albeit lacking the N-terminal sequence of a functional core protein, HGV builds classical viral particles displaying E2 envelope protein on their outer surfaces.
    Journal of Virology 06/1998; 72(5):4541-5. · 5.08 Impact Factor
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    ABSTRACT: The second envelope protein (E2) of the hepatitis G virus (HGV) was expressed in Chinese hamster ovary (CHO) cells and showed a molecular weight of approximately 60 to 70 kd, with 15 to 25 kd of the size contributed by N-linked glycosylation. An enzyme-linked immunosorbent assay (ELISA) using HGV-E2 was developed to test for antibodies to this protein (anti-E2) in human sera. High sensitivity was achieved by developing monoclonal antibodies (mAbs) to HGV-E2, which were used as capture antibodies in the ELISA. Our studies revealed that 16% of healthy Spanish blood donors were exposed to HGV, indicating that additional routes of viral transmission besides parenteral exposure might exist. An even higher prevalence of exposure to HGV (52%-73%) was found in several groups at risk of parenteral exposure to infectious agents, i.e., intravenous drug users, transfusion history, hemophiliacs, and hepatitis C virus (HCV)-positive patients. Most anti-E2-positive patients were HGV-RNA-negative and vice versa, indicating an inverse correlation of these two viral markers. A panel of 16 posttransfusion patients followed for up to 16 years revealed that patients who develop an anti-E2 response become HGV-RNA-negative, while patients who do not develop anti-E2 are persistently infected. Immunity to HGV seems to be long-lasting, because circulating antibody to E2 could still be detected 14 years after seroconversion. Sequence comparisons showed that E2 is highly conserved among isolates collected worldwide, indicating that immune escape variants are not common in HGV infections. This reflects on a molecular level why HGV infections usually are cleared spontaneously by the host. However, possible mechanisms of HGV persistence, as found in some patients, remain to be elucidated.
    Hepatology 01/1998; 26(6):1626-33. · 12.00 Impact Factor
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    ABSTRACT: We reported previously on an area in Japan where over 30% of the inhabitants were positive for hepatitis C virus (HCV) antibody. In the present study, clinical features of hepatitis G virus (HGV) infection in this area of high endemicity were compared to those in an area where HCV is not endemic. A total of 400 individuals were selected randomly from those who were medically screened for liver disease in 1993; 200 were from the high-endemicity area, and the other 200 were from the no-endemicity area. HGV RNA was measured by reverse transcription and PCR with primers in the 5' noncoding region. Antibody to HGV envelope protein E2 was measured by an enzyme-linked immunosorbent assay. Prevalence of any HGV marker in the high-endemicity area (32%) was significantly (P < 0.0001) higher than that in the no-endemicity area (6%); similar differences, 32% versus 3% (P < 0.0001), had been observed for HCV markers (HCV RNA and HCV antibody). In areas of both high and no endemicity, HCV markers were significantly more prevalent in individuals with any HGV marker than in those without HGV markers, and age-specific prevalence of HGV markers was distributed similarly to that of any HCV marker. Among possible routes of HGV transmission that were analyzed, folk medicine was significant in the high-endemicity area, but blood transfusion was the major route in the no-endemicity area. The rate of accompanying viremia in HGV infection (15%) was significantly lower than that in HCV infection (78%) (P < 0.0001). In conclusion, HGV infection was highly prevalent in the area of high HCV endemicity and was closely associated with HCV infection. HGV seemed to be transmitted via the practice of folk medicine as well as blood transfusion. HGV resulted in a chronic carrier state less frequently than did HCV.
    Journal of Clinical Microbiology 01/1998; 36(1):110-4. · 4.07 Impact Factor
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    ABSTRACT: Hepatitis G virus (HGV/GBV-C) RNA indicating current infection has been frequently isolated from the sera of transplant recipients and other multitransfused individuals. Lifetime exposure to the virus, however, is unknown. We carried out a study to determine the prevalence and risk factors of HGV antibodies and of HGV RNA among renal transplant recipients, and to investigate possible associations between HGV RNA and immunosuppressive treatment. HGV RNA was detected by reverse transcriptase-polymerase chain reaction, and HGV antibodies (anti-E2) by a newly developed immunoassay. To assess risk factors for HGV exposure, univariate and multivariate analysis was performed. Of the 221 patients, 14% were HGV RNA positive and 40% had HGV antibodies. Both HGV RNA and anti-HGV were present in only two individuals. Thus, the overall HGV exposure prevalence was 53%. It increased significantly with the number of blood transfusions. In logistic regression, the adjusted HGV exposure prevalence odds ratio was 5.7 (95% confidence interval [CI]: 2.2-15) among patients with > or =10 transfusions (baseline: no transfusions). Other independent risk factors were a longer duration of hemodialysis and a longer time interval since transplantation. HGV viremia was not associated with the type of immunosuppressive treatment. Alanine aminotransferase levels were not significantly increased among HGV RNA-positive patients. Much higher proportions of renal transplant recipients were exposed to HGV than is suggested by HGV RNA detection alone. The majority of infected individuals apparently eliminate the virus over time. Contaminated blood transfusions have to be regarded as a main risk factor for HGV infection.
    Transplantation 08/1997; 64(4):608-12. · 3.78 Impact Factor
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    ABSTRACT: A flavivirus designated hepatitis G virus (HGV) has been isolated from the serum of patients with non-A-E hepatitis. Hitherto, the presence of HGV RNA in serum has been detected with the reverse transcription-polymerase chain reaction (RT-PCR) amplification method. We have now developed an immunoassay for antibodies against an HGV protein. Recombinant HGV envelope protein E2 was used as antigen in an ELISA. 80 blood donors, 99 intravenous-drug users, and 11 patients with acute post-transfusion hepatitis were tested for antibodies to E2. The HGV-RNA status was assessed by RT-PCR. Anti-E2 seroprevalence was 9% among the blood donors and 41% among the drug users; HGV-RNA prevalence was 2.5% and 38%, respectively. Whereas anti-E2 prevalence increased with the duration of drug use, HGV-RNA prevalence declined in parallel. In each group, the presence of anti-E2 and HGV RNA was almost mutually exclusive: none of the blood donors and only 4% of the drug users were positive for both markers at the same time. Of the 11 post-transfusion patients--who were all HGV-RNA positive and anti-E2 negative at the onset of disease--four developed antibodies to E2 during the following year, and two of the four subsequently became HGV-RNA negative. We conclude that a humoral immune response to E2 is associated with loss of detectable HGV viraemia. Thus, E2-specific antibodies might serve as a useful marker for diagnosing recovery from HGV infections. The immunoassay we describe should facilitate investigation of suspected infections and may be helpful in the elucidation of the clinical significance of HGV.
    The Lancet 03/1997; 349(9048):318-20. · 39.21 Impact Factor
  • Immunology Letters - IMMUNOL LETT. 01/1997; 56:296-296.
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    ABSTRACT: The recently identified hepatitis G virus (HGV) is parenterally transmitted; the impact of sexual transmission is unknown. Moreover, it is unclear what proportion of HGV-infected persons may develop persistent viremia. Sera from injecting drug users (IDUs), non-drug-injecting homosexual and bisexual men with high levels of sexual risk behavior, and blood donors were tested for HGV RNA and hepatitis C virus (HCV) RNA by reverse transcriptase-polymerase chain reaction and for antibodies to human immunodeficiency virus, hepatitis B virus, and HCV. HGV RNA was detected in 33% of IDUs (n = 130), 11% of homosexual and bisexual men (n = 101), and 2% of blood donors (n = 90). HGV RNA seroprevalence significantly decreased with increasing time since first drug injection, whereas the seroprevalences of both HCV RNA and anti-HCV antibody increased. Thus, a high proportion of HGV-infected persons may clear the virus and develop protective antibodies. The relatively high HGV RNA prevalence among non-drug-injecting homosexual and bisexual men indicates that sexual contact may be another important route of HGV transmission.
    The Journal of Infectious Diseases 01/1997; 174(6):1320-3. · 5.85 Impact Factor
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    ABSTRACT: Hepatitis G virus (HGV) was recently identified as a new member of the Flaviviridae, but its clinical significance is still unclear. Since no immunoassay for the diagnosis of HGV is available, we developed a sensitive reverse transcription-PCR (RT-PCR) assay to facilitate the detection of the viral genome by mass screening in the clinical laboratory. Sequences within the 5'-noncoding region and within the putative NS5a region are independently amplified in the presence of digoxigenin-11-dUTP and are detected by hybridization with biotinylated capture probes binding to a streptavidin-coated matrix. Semiquantitative Enzymun-Test DNA detection via chemiluminescence can be performed either in a microtiter plate format or on fully automated ES 300 machines. We were able to detect at least 8 x 10(2) genome equivalents per ml of serum using both primer pairs. HGV was shown to be present in 43 of 130 (33%) serum samples from intravenous drug abusers with a high risk of parenteral exposure. However, only two of the patients were positive when the NS5a primers only were used, and only one patient was positive when only the 5'-noncoding region primers were used, demonstrating the increased sensitivity of HGV detection with two sets of primers. Among these patients, there was no obvious correlation with other viral infections like hepatitis B virus, hepatitis C virus, or human immunodeficiency virus. Within a blood donor panel, 3 of 92 (3%) samples were found to be HGV positive, suggesting that donated blood may need to be screened for HGV.
    Journal of Clinical Microbiology 12/1996; 34(11):2660-4. · 4.07 Impact Factor
  • Journal of Microbiological Methods 09/1996; 27(1):98-99. · 2.16 Impact Factor
  • Journal of Microbiological Methods - J MICROBIOL METH. 01/1996; 27(1):99-99.
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