Angela Uy

Association of hepatitis C virus in human sera with β-lipoprotein

Hepatitis C virus (HCV)-RNA in sera of patients with viral hepatitis C is supposed to be included, at least partially, into HCV particles. We found that the density of HCV-RNA-carrying material was variable, as determined by sucrose gradient density centrifugation (1.03–1.20 g/cm3). In some of the sera examined HCV-RNA was restricted to low densities between 1.03 and 1.08 g/cm3. In other sera additional densities of HCV-RNA were found distributed over the whole gradient with peaks at 1.12 and 1.17 and at 1.19–1.20 g/cm3. HCV-RNA banding at low densities could be completely co-precipitated with anti-β lipoprotein, whereas HCV-RNA fractions of higher densities were only partially precipitated or not at all. In 8 of 20 sera directly examined, HCV-RNA could be completely and in 9 sera only partially co-precipitated by anti-β lipoprotein. In 3 sera no significant precipitation could be observed.

Precore sequence of hepatitis B virus inducing e antigen and membrane association of the viral core protein

Hepatitis B virus (HBV) DNA contains a precore (pre-c) sequence of 29 codons with unknown function upstream of its gene for the major core protein. Its significance was studied by expression of core proteins with and without pre-c in Escherichia coli. Core protein without pre-c, P22c, assembled spontaneously to core particles and formed core antigen. It had the same size and antigenicity as core particles from infected liver. Core protein with pre-c, P25e, instead formed membrane-associated e antigen (HBeAg). The data suggest that pre-c functions as a signal peptide for the attachment of core protein P25e to cellular membranes. This hypothesis can explain the not yet understood relation between viremia and HbeAg and the protective role of anti-HBe antibody.

Assay of hepatitis B viras genome titers in sera of infected subjects

A method for quantitative standardization of the DNA hybridization assay for hepatitis B virus (HBV) DNA protein complex in serum is described. This method was used to determine the titer of HBV DNA in various groups of subjects with HB surface antigen (HBsAg) in order to ascertain its accuracy as an index of infectivity. The methods detection limit was 105 genome equivalents or 0.3 pg DNA per ml. Titers of 5×107 to 5×108 genome equivalents per ml were found to be typical for persistent massive viremia, which occurred more frequently in symptomatic (30 of 48) than in asymptomatic (24 of 72) carriers positive for HBe antigen (HBeAg). Moderate viremia (10s5−5×107) was usually found in patients eliminating the virus from the blood. Patients with resolving acute hepatitis B were frequently positive at the onset (18 of 26) with moderate titers, but became negative within several weeks. In 11 patients who developed chronic hepatitis B, titers increased until typical massive viremia was evident. Whereas healthy HBsAg carriers with anti-HBe always had negative genome titers (144 of 144), symptomatic carriers with anti-HBe often had moderate genome titers (9 of 30). It is recommended that genome titers be monitored in HBeAg-positive and in symptomatic anti-HBe positive virus carriers in order to distinguish between virus carriers with high (>5×107), moderate (105−5×107) and low (<105) infectivity.

Testing of individual blood donations for HCV RNA reduces the residual risk of transfusion-transmitted HCV infection.

BACKGROUND: To allow cost‐effective RNA testing with NAT techniques, the national authorities of several countries have planned or already introduced tests of mixed specimens, that is, plasma pools.

Molecular epidemiology of an outbreak of HCV in a hemodialysis unit: direct sequencing of HCV-HVR1 as an appropriate tool for phylogenetic analysis.

Infection with hepatitis C virus (HCV) is still a serious problem in hemodialysis patients, despite screening of blood products for anti‐HCV antibodies. The prevalence of HCV in HD patients is between 15% and 30% in Germany. We report the molecular epidemiology of an HCV outbreak in a hemodialysis unit in 1997 is determined. HCV hypervariable region 1 (HVR1) was amplified from serum samples of 19 patients by polymerase chain reaction (PCR) and sequenced directly. In addition, HCV isolates from 3 of these 19 patients were cloned and sequenced. 14 newly infected patients and two patients, who had been infected for several years had very closely related HCV isolates. Unrelated HCV isolates as well as sequences obtained from an HCV outbreak in a plasmapheresis center were found in different, distantly related branches. These findings provide strong evidence for nosocomial transmission of the virus, despite following strict general hygiene precautions. The production of anti‐HCV antibody was delayed significantly or seroconversion did not occur at all during the period of observation in 8 out of 14 newly infected HCV RNA positive patients. Close‐meshed reverse transcription‐polymerase chain reaction (RT‐PCR) analyses on apparently non infected patients within hemodialysis units and upon admission of new patients is strongly recommended for the early detection and prevention of outbreaks of HCV. J. Med. Virol. 60:152–158, 2000.

Genomic variability in the preS1 region and determination of routes of transmission of hepatitis B virus

On the basis of published sequence data the preS1 attachment region of hepatitis B virus (HBV) appears to be highly variable. Using a novel method for rapid DNA sequencing by the polymerase chain reaction we screened 34 HBV DNA-positive sera for mutations in a variable part of the preS1 region of the HBV genome. The sequence data were used to analyse potential chains of infection, and strongly supported the expected routes of HBV transmission among patient groups. Furthermore, sequence comparisons permitted sub-genotyping of the viruses. In the 22 cases of subtype adw, we found a very low number of point mutations. This shows that the attachment site of HBV is more highly conserved than that of other blood-transmissible viruses such as human immunodeficiency virus or hepatitis C virus.

Risk of hepatitis C virus (HCV) transmission by anti-HCV-negative blood components in Austria and Germany

In order to estimate the residual risk of transfusion-transmitted HCV infection, we have analyzed data from two transfusion centers in Austria (Vienna) and Germany (G6ttingen) from 1990 to 1995. In Vienna, the seroprevalence (RIBA-confirmed third-generation anti-HCV tests) was 0.28% in first-time donors (FTD) and the incidence of seroconversion in repeat donors (RD) was 0.049 (per 100 person years) from 1994 to 1995. In Göttingen, the prevalence of a PCR-confirmed positive third-generation anti-HCV test was 0.22% in FTDs and the incidence was 0.093 (per 100 person years). A continuous decline of the rate of anti-HCV-positive donations and donors was observed with first- and second-generation anti-HCV tests in the years 1990–1994. The introduction of the third-generation anti-HCV test resulted in increased numbers of anti-HCV positive repeat donors, mainly due to false-positive results. Only 9% of anti-HCV-positive repeat donors were either PCR positive or RIBA positive or indeterminate. Based on a mathematical model which takes (a) the window period, (b) the false-negative rate of anti-HCV tests, and (c) human and operational errors into consideration, we have calculated the residual risk of HCV infection. We used a window period of 74 days, a sensitivity of 98%, and an error rate of 0.1%. The residual risk (for third-generation anti-HCV test-negative blood components) was calculated to be 1:9000 (95% confidence interval 1:16390–1:6210) and 1:4800 (95% confidence interval 1:40000–1:1320) for Vienna and Göttingen, respectively, in 1994 and 1995. Since this conservative approach does not take the impact of ALAT screening into account, the actual risk is probably lower.

Assay of preS epitopes and preS1 antibody in hepatitis B virus carriers and immune persons

The diagnostical significance of the large hepatitis B surface protein with its preS1 attachment site and of anti-preS antibodies are not yet well known. We investigated the epitope of the preS1 attachment site to see whether it is a marker of viremia and whether antibodies against it occur in convalescents and vaccinees. For comparison, sera were also tested for the presence and relative amount of a preS2 epitope. The epitopes were detected by binding to specific monoclonal antibodies (mAb MA18/7 for the preS1 epitope and mAb Q19/10 for the preS2 epitope) at the solid phase of a sandwich enzyme-linked immunosorbent assay. Antibody against the preS1 epitope was detected by inhibition of binding to mAb MA18/7. This mAb inhibits attachment of preS1 antigen to hepatocytes and reacts with a subtypeindependent sequential epitope at the surface of hepatitis B virus between amino acid 29–36. This preS1 epitope occurs in most hepatitis B surface antigen (HBsAg) carriers, irrespective of viremia. Free preS2 epitope Q19/10 is present in samples with more than 8 μg/ml total HBsAg and it is masked in sera with less HBsAg. Antibodies which compete with mAb MA18/7 for its viral preS1 epitope occur in one third of HBsAg carriers who were negative for hepatitis B e antigen. It also occurs in one third of convalescents and in most good responders to plasma-derived vaccines.

Haemolysis in hepatitis A virus infections coinciding with the occurrence of autoantibodies against triosephosphate isomerase and the reactivation of latent persistent Epstein-Barr virus infection.

Haemolysis has been observed frequently as a complication of acute hepatitis A virus (HAV) infection. However, the pathogenic mechanism has not been elucidated completely. In individual cases the detection of anti‐erythrocyte antibodies of unknown specificity was described. The raised serum IgM fraction was shown to consist partially of autoantibodies. Previously, we detected autoantibodies of immunoglobulin class M directed against triosephosphate isomerase (IgM anti‐TPI) in patients with infectious mononucleosis. These autoantibodies are able to induce haemolysis.

Hemolysis and Autoantibodies to Triosephosphate Isomerase in a Patient with Acute Hepatitis A Virus Infection

Having returned from a holiday in Southeast Europe, a 30-year-old German woman developed acute hepatitis. Hepatitis A virus (HAV) infection was diagnosed serologically. During the course of the infection, hemolysis was found. IgM antibodies against triosephosphate isomerase (IgM anti-TPI) were detected in the patients serum from the acute phase of the HAV infection. Affinity purified IgM anti-TPI from the serum reduced the enzyme activity in vitro and caused an increased 51Cr release from erythrocytes. IgM anti-TPI is assumed to be one of the causative agents of hemolysis in HAV infection.