R. Thomssen

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.

Low density lipoprotein receptor as a candidate receptor for hepatitis C virus

Hepatitis C virus (HCV) binds to different human cell lines in vitro. However, the efficiency of adsorption is very low due mainly to a relatively small fraction of the virus being able to bind to these cells. Free low density lipoprotein (LDL > 200 μg/ml) is able to block the attachment of HCV to human fibroblasts in vitro completely. COS‐7 cells being primarily not able to bind HCV were transfected with a vector containing the entire coding sequence of the human LDL‐receptor (LDLR). HCV was now bound to these cells. We propose that HCV and LDL are competitive for the cellular LDLR and that LDL in sera of patients may regulate the binding of HCV to this target. J. Med. Virol. 57:223–229, 1999.

Density heterogeneities of hepatitis C virus in human sera due to the binding of β-lipoproteins and immunoglobulins

Heterogeneities in the density of hepatitis C virus RNA-carrying material (HCV-RNA-CM) found in human sera (1.03–1.20 g/cm3) are attributed to the binding of low-density lipoproteins and/or of IgG. In some sera HCV-RNA-CM seems to be nearly totally bound to β-lipoproteins and cannot be precipitated by anti-IgG (γ); in others more than 95% of HCV-RNA-CM is bound to IgG and cannot be precipitated by anti-β-lipoprotein. Furthermore, there are sera from which HCV-RNA-CM can be completely be precipitated by either anti-β-lipoprotein or anti-IgG (γ), pointing to a binding of the two serum proteins to the same HCV-RNA-CM. There are other sera from which HCV-RNA-CM can be partially precipitated by the one or the other antiserum, leaving behind fractions, which are bound to β-lipoprotein or to IgG. HCV-RNA-CM cannot be precipitated from some sera either by anti-β-lipoprotein or by anti-IgG (γ).

Epidemiology and Clinical Manifestations of Lyme Borreliosis in Childhood.: A Prospective Multicentre Study with Special Regard to Neuroborreliosis

ABSTRACT. Lyme borreliosis is a tick‐borne infection caused by the spirochete Borrelia burgdorferi, whose discovery in 1982 solved an aetiological mystery involving a variety of dcrmatological and neurological disorders and explained their association with Lyme disease. Lyme borreliosis occurs frequently and is readily treatable with antibiotics.

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.

Binding of human lipoproteins (low, very low, high density lipoproteins) to recombinant envelope proteins of hepatitis C virus

Abstract Heterogeneities in the density of hepatitis C virus (HCV)-RNA-carrying material from human sera (1.03–1.20 g/ml) are partially due to the binding of lipoproteins [low density (LDL), very low density (VLDL), high density (HDL) lipoproteins] and immunoglobulins. In this study we demonstrate the binding of recombinant HCV envelope protein (E1/E2) to human LDL, VLDL and HDL on a molecular basis. The binding of lipoproteins was restricted to the middle part of the E1 gene product (amino acids 222–336) and the C-terminal part of the E2 protein (amino acids 523–809). Lipoproteins did not bind to recombinant HCV core protein.

Hepatitis B Virus Core Gene Mutations Which Block Nucleocapsid Envelopment

ABSTRACT Recently we generated a panel of hepatitis B virus core gene mutants carrying single insertions or deletions which allowed efficient expression of the core protein in bacteria and self-assembly of capsids. Eleven of these mutations were introduced into a eukaryotic core gene expression vector and characterized by transcomplementation of a core-negative HBV genome in cotransfected human hepatoma HuH7 cells. Surprisingly, four mutants (two insertions [EFGA downstream of A11 and LDTASALYR downstream of R39] and two deletions [Y38-R39-E40 and L42]) produced no detectable capsids. The other seven mutants supported capsid formation and pregenome packaging/viral minus- and plus-strand-DNA synthesis but to different levels. Four of these seven mutants (two insertions [GA downstream of A11 and EHCSP downstream of P50] and two deletions [S44 and A80]) allowed virion morphogenesis and secretion. The mutant carrying a deletion of A80 at the tip of the spike protruding from the capsid was hepatitis B virus core antigen negative but wild type with respect to virion formation, indicating that this site might not be crucial for capsid-surface protein interactions during morphogenesis. The other three nucleocapsid-forming mutants (one insertion [LS downstream of S141] and two deletions [T12 and P134]) were strongly blocked in virion formation. The corresponding sites are located in the part of the protein forming the body of the capsid and not in the spike. These mutations may alter sites on the particle which contact surface proteins during envelopment, or they may block the appearance of a signal for the transport or the maturation of the capsid which is linked to viral DNA synthesis and required for envelopment.

PERIPHERAL FACIAL PALSY IN CHILDHOOD - LYME BORRELIOSIS TO BE SUSPECTED UNLESS PROVEN OTHERWISE

ABSTRACT. 27 consecutive cases with acute peripheral facial palsy were studied for Lyme borreliosis. In 16 out of 27 children Lyme borreliosis could be diagnosed by detection of specific IgM antibodies in CSF. CSF findings allow a clear distinction according to etiology. All children with facial palsy due to Lyme borreliosis revealed lymphocytic CSF pleocytosis, whereas in cases of unknown etiology CSF was usually normal. Bilateral facial palsy occurred only in children with Lyme borreliosis. All cases with a positive history of tick bite and/or erythema migrans in the head‐neck region showed ipsilateral neurological affection suggesting a direct invasion via the affected nerve by Borrelia burgdorferi.

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.

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.