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ABSTRACT: Standard preparations for HBsAg are required for quality control of test kits and clinical studies on HBsAg quantitation. WHO provides purified heat inactivated HBsAg diluted in negative defribinated plasma as 2nd International Standard (IS) for quality control of tests.
Study of possible alterations of antigenicity, protein composition, size and density of the heat inactivated source material (SM) for the 2nd IS.
Native HBsAg and SM were examined by quantitative immune electrophoresis (QIE), SDS-PAGE, ultracentrifugation and gel chromatography. HBV DNA was sequenced and the HBsAg geno/subtype derived.
The SM contained 97,600 International Units HBsAg/ml in QIE which agreed very well with the previous evaluations by WHO using 10 different assays. In SDS-PAGE, SM showed on a strong background the small HBs proteins but no preS proteins. SM had a more heterogeneous density than native HBsAg and contained particle aggregates. The HBsAg geno/subtype of SM was A2/adw2.
The IS has very good HBs antigenicity, but it lacks the preS domains, has modified HBs proteins and is partially aggregated. While it has been proven very useful for quality control of tests, certain inconsistencies due to the altered structure of its HBsAg cannot be excluded.
Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 03/2010; 47(3):238-42. · 3.12 Impact Factor
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ABSTRACT: The Taormina Consensus Conference defined 'occult hepatitis B virus (HBV) infection' (OBI) as the 'presence of HBV DNA in the liver of individuals testing HBsAg-negative with currently available assays'. Most occult is the so-called 'window period' after exposure before HBV DNA appears in the blood. We identified two blood donors whose donations tested HBsAg- and HBV DNA-negative, but transmitted HBV. Both subsequently developed HBsAg and acute hepatitis. However, such cases are not considered as true OBI. A true transient OBI remains HBsAg-negative during the entire course. One case of acute OBI showed a peak viremia of 15,000 IU/ml HBV DNA and sub-borderline HBsAg, suggesting a ratio of virions to subviral particles of 1:10, whereas 'normal' cases show at peak viremia a ratio of 1:3,000. Blood donors with OBI may transmit HBV. We studied 5 blood donors with OBI and 55 of their recipients. In 22 recipients, transmission was probable, but they remained healthy. However, in 3 recipients, who were immunosuppressed at the time of transfusion, fatal fulminant hepatitis B developed. The majority of anti-HBc-positive healthy individuals have HBV DNA in the liver which may start replication under severe immunosuppression. Nine such cases are described here. OBI or reactivated HBV infections often lead to selection of HBsAg escape mutations as we could show in 11 of 14 cases. Infection of vaccinated individuals favors development of OBI as we observed in 6 blood donors. HB vaccination may solve the problem of overt HBV infection but may favor OBI.
Digestive Diseases 01/2010; 28(1):116-25. · 2.37 Impact Factor
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ABSTRACT: Core antigen (HBcAg) is the most immunogenic component of hepatitis B virus (HBV) and is believed to induce virtually always antibodies (anti-HBc) in immunocompetent infected persons. However, some chronically infected persons do not develop detectable anti-HBc.
A more sensitive assay for anti-HBc was to be developed and used to re-evaluate a cohort of chronically HBV infected persons without detectable anti-HBc.
Among 3309 serum samples which had been tested by commercially available (microparticle) enzyme immune assay (M/EIA) 34 samples from 22 patients were identified having reacted positive for HBsAg and negative for anti-HBc. Nine of these patients had immunosuppression or HIV coinfection, 13 patients were immunocompetent, 5 of them were perinatally infected. Anti-HBc was re-tested for in an immune precipitation (IP) assay using (32)P-labelled recombinant HBcAg as reagent and anti-human-IgG-coated magnetic beads as separation system for immunecomplexes containing HBcAg. Specificity was controlled for by competition with unlabelled HBcAg.
27 serum samples from the 22 patients could be retested. IP was positive in 7 MEIA negative sera, unspecific positive in 4 and negative in 16. Using 5 anti-HBe positive control sera, we found IP to be 1.8-fold (1.3-2.9) more sensitive than MEIA, but IP was 6.5-fold (5.8-7.4) more sensitive with 4 anti-HBe negative, anti-HBc positive sera.
IP allowed specific detection of anti-HBc in about 25% of MEIA negative chronic HBV patients. The majority of these seem to produce no or very little anti-HBc, however.
Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 08/2009; 46(2):124-8. · 3.12 Impact Factor
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ABSTRACT: Hepatitis C virus (HCV) is a positive strand RNA virus that propagates primarily in the liver. We show here that the liver-specific microRNA-122 (miR-122), a member of a class of small cellular RNAs that mediate post-transcriptional gene regulation usually by repressing the translation of mRNAs through interaction with their 3'-untranslated regions (UTRs), stimulates the translation of HCV. Sequestration of miR-122 in liver cell lines strongly reduces HCV translation, whereas addition of miR-122 stimulates HCV translation in liver cell lines as well as in the non-liver HeLa cells and in rabbit reticulocyte lysate. The stimulation is conferred by direct interaction of miR-122 with two target sites in the 5'-UTR of the HCV genome. With a replication-defective NS5B polymerase mutant genome, we show that the translation stimulation is independent of viral RNA synthesis. miR-122 stimulates HCV translation by enhancing the association of ribosomes with the viral RNA at an early initiation stage. In conclusion, the liver-specific miR-122 may contribute to HCV liver tropism at the level of translation.
The EMBO Journal 12/2008; 27(24):3300-10. · 9.20 Impact Factor
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Wolfram H. Gerlich,
Franz F. Wagner,
Michael Chudy,
Lene Holm Harritshoj,
Annette Lattermann,
Sandra Wienzek,
Dieter Glebe,
Mona Saniewski, Christian G. Schüttler,
Ulrike C. Wend,
Wulf R. Willems,
Ursula Bauerfeind,
Christine Jork,
Gregor Bein,
Per Platz,
Henrik Ullum,
Ebbe Dickmeiss
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ABSTRACT: Five blood donors with occult persistent and one donor with an early window phase HBV infection were identified. They were negative in regular HBsAg screening and had low levels of HBV DNA that were probably not detectable by current mini-pool nucleic acid amplification testing. In four donors several mutations were found located in the HBs antigen loop. In three donors the mutations were predominantly outside the “a” determinant; one donor had a wild type HBsAg sequence. Fifty-five recipients of donations from the persistently infected donors could be tested for previous or ongoing HBV infection and of them 53% (29/55) were anti-HBc positive. Based on the prevalence of anti-HBc in Germany (7%), it was assumed that four of those recipients had already been positive before transfusion. In 22 cases, it was assumed that they acquired infection by the donations, but the infection remained asymptomatic and was resolved. In three cases transmission was proven by the time course of the acute infection and sequence identity The resulting infection was fatal and associated with immunological disorders at the time of transmission: in one case sepsis and in the other two cases immunosuppression. In a further asymptomatic case of proven transmission from the early window phase donation passively administered anti-HBs could not prevent spread of wildtype HBV but antiviral treatment lead to resolution. Surprisingly the typical acute hepatitis B was not observed in a single one of the 26 cases of assumed or proven transmission. J. Med. Virol. 79:S32–S36, 2007. © 2007 Wiley-Liss, Inc.
Journal of Medical Virology 12/2006; 79(S1):S32 - S36. · 2.82 Impact Factor
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ABSTRACT: Quantification of hepatitis C virus (HCV) core antigen and RNA in serum samples leads to a highly variable ratio of both. It is not clear whether this is due to the inaccuracy of RNA quantification or whether both are independent parameters in a certain range. We established a real-time reverse transcription (RT)-PCR for HCV RNA that combines very high sensitivity with a large dynamic range and minimal standard deviations. The assay was calibrated with the first international standard, 96/790, and the international genotype panel for HCV from the National Institute of Biological Standardisation and Control. A linear readout was obtained between 200 and 5 x 10(7) IU/ml. The detection limit was 80 IU/ml, the reproducibility was <0.05 log, and the standard error within one run was <0.01. Comparison of the method with the Roche Monitor competitive RT-PCR revealed its high accuracy. The core protein concentration was determined within a range from 1.5 to 400 pg/ml by using the preliminary trak-C assay from Ortho Clinical Diagnostics. Correlating the HCV RNA levels with core antigen concentrations in 197 serum samples from 23 interferon-treated patients, a average ratio of 7,900 IU of HCV RNA per pg of core antigen was estimated, but the variability of this ratio exceeded largely the variability of the two assays, ranging from 50 to 20,000 IU/pg. Theoretically, HCV should contain ca. 43,000 IU of RNA/pg core. In conclusion, the core antigen assay seems to detect, in addition to complete virions, RNA-free core protein structures, which enhances its sensitivity (98% in this group). The variable ratio of RNA and core protein is not mainly due to standard deviations of quantification but could be an additional parameter for treatment follow-up and state of viral replication.
Journal of Clinical Microbiology 06/2004; 42(5):1977-81. · 4.15 Impact Factor
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ABSTRACT: Epidemiological studies have shown that coinfection or superinfection with hepatitis B virus (HBV) and C virus (HCV) frequently leads to the suppression of hepatitis B virus replication. The mechanism of this phenomenon is still unclear. Shih et al. [J Virol 1993;67:5823] reported a direct suppression of HBV replication by the core protein of HCV. The target structure of HCV core protein in this system remained unclear.
As HCV core protein has been shown to influence expression from transcriptional elements, we studied whether HCV core protein altered the activity of the two HBV enhancers 1 and 2. Luciferase vectors for HBV enhancers 1 or 2 were cotransfected with expression constructs for HCV core protein in murine and human hepatocyte lines.
Full-length HCV core protein suppressed the HBV enhancer 1 up to 11-fold, the enhancer 2 3-4-fold. Suppression of HBV enhancer 1 by HCV core from genotype 1b was stronger than by HCV core of genotypes 3a or 1a. Carboxyterminally truncated core proteins had lower or no suppression activity.
These data suggest that HCV core protein may directly repress transcription of the HBV RNAs. This trans-repression may contribute to suppression of HBV replication in patients coinfected with both viruses.
Journal of Hepatology 01/2003; 37(6):855-62. · 9.26 Impact Factor
