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Biomedical subjects

G Siegl

Publications and source records attributed to G Siegl.

At least 55 records · Page 3Linked to original sources
PubMed1990

Serodiagnosis of infectious mononucleosis by using recombinant Epstein-Barr virus antigens and enzyme-linked immunosorbent assay technology.

Four recombinant, diagnostically useful Epstein-Barr virus (EBV) proteins representative of the viral capsid antigen (p150), diffuse early antigen (p54), the major DNA-binding protein (p138), and the EBV nuclear antigen (p72) (W. Hinderer, H. Nebel-Schickel, H.H. Sonneborn, M. Motz, R. Kühbeck, and H. Wolf, J. Exp. Clin. Cancer Res. 7[Suppl.]:132, 1988) were used to set up individual enzyme-linked immunosorbent assays (ELISAs) for the qualitative and quantitative detection of immunoglobulin M (IgM) and IgG antibodies. In direct comparison with results obtained by standard immunofluorescence or immunoperoxidase assays, it was then shown that the recombinant EBV ELISAs provide the means for specific and sensitive serodiagnosis of infectious mononucleosis (IM) caused by EBV. The most useful markers in sera from such patients proved to be IgM antibodies against p54, p138, and p150. Additional positive markers for recent or ongoing IM apparently were IgG antibodies against p54 and p138. In contrast, anti-p72 IgG had a high preference for sera from healthy blood donors and, therefore, can be considered indicative of past exposure to the virus. Altogether, the individual ELISAs proved to be as specific and at least as sensitive for the diagnosis of IM as the currently available standard techniques are. Moreover, our findings suggest that, by combining individual test antigens, a workable ELISA system consisting of three assays (IgM against p54, p138, and p150; IgG against p54 and p138; and IgG against p72) can be established for the standardized rapid diagnosis of acute EBV infections.

Adolescent↗
PubMed1990

Molecular biology and pathogenicity of human and animal parvoviruses.

The so-called autonomous parvoviruses comprise a wide spectrum of viral agents with quite similar physicochemical and biological properties. Characteristically, they are small, naked, and extraordinarily stable particles made up of only 2-4 structural proteins and a genome of linear, single-stranded DNA consisting of 4500-5600 nts. For replication they depend on cellular helper functions which become available at distinct stages during cellular differentiation (developmentally-dependent functions) and transiently during the phase of DNA synthesis in the mitotic cycle (cell cycle-dependent function(s)) of a susceptible cell. Most of the clinical syndromes induced by parvoviruses therefore can be related to infection of mitotically active cells or tissues at different stages in the development and maturation of the host organism. This view of parvovirus pathogenicity holds true for all animal parvoviruses as well as for the human parvovirus B19.

Animals↗
PubMed1989

Detection of defective genomes in hepatitis A virus particles present in clinical specimens.

Hepatitis A virus (HAV) particles harbouring a physically defective RNA genome have been reported to occur in all HAV-infected cell culture systems analysed so far. The most prominent defects consist of three distinct overlapping deletions in the region of the HAV genome encoding the structural proteins. By probing for the endpoints of these deletions in RNA samples using S1 nuclease and exonuclease VII mapping, we obtained suggestive evidence for the existence also of defective genomes in HAV particles present in faecal specimens, in viraemic blood collected in the course of hepatitis A virus infection in man, as well as in the liver of an experimentally infected marmoset monkey. The deletions identified extend from nucleotide (nt) 1200 to nt 3820 and from nt 1200 to nt 3240 of the HAV genome. They are compatible with two of the deletions detected in particles grown in vitro in cell cultures and shown to interfere with the replication of standard hepatitis A virions.

Animals↗
PubMed1988

[Parvoviruses as pathogenic agents in man].

Since 1981 an increasing number of human parvovirus infections have been reported. There is now clear evidence that parvovirus B19 is the causative agent of erythema infectiosum (fifth disease). Further clinical situations associated with this virus include aplastic crisis in patients with hemolytic anemia and acute and persistent arthropathies preferentially occurring in adult women. Transplacental infection during pregnancy may result in hydrops foetalis, intrauterine death and spontaneous abortion. Additional, thus far barely characterized parvoviruses have been isolated from individuals with enteritis or rheumatoid arthritis. The present review summarizes today's knowledge of the biological properties and relevance of human parvoviruses as pathogens. The potentialities and limitations of laboratory diagnosis in parvovirus infections are discussed.

Anemia, Hemolytic↗
PubMed1988

Detection and characterization of subgenomic RNAs in hepatitis A virus particles.

Defective viral particles containing deleted genomes were detected in harvests of cell cultures infected with various HAV isolates. The most prominent deletions were identified within the region of the genome coding for structural proteins. In this location three different deletions spanning nts 930-4380 (A), 1140-3820 (B), and 1370-3240 (C) were characterized. In addition to these internal deletions, various truncated RNAs were detected lacking either partially or completely the 3' terminal region which is supposed to code for viral replicase. RNA molecules with internal deletions as well as those with 3' terminal truncations could also be extracted directly from infected cells. During multiple consecutive passages of a given HAV strain, deletions A, B, and C accumulated and a quantitative increase of deleted RNAs occurred. Type and predominance of deletions varied with virus strains (CLF, GBM, MBB11/5, HM175, CR326, H141) and with the type of cells used for propagation (MRC-5, BGM, HELF, PLC/PRF/5). However, within the limits of the reliability of S1 analysis the endpoints of deletions A, B, and C were conserved. The mechanisms leading to formation of deletions remain unclear. Yet, some sequences flanking internal deletions showed homology with common splice signals and 3' terminal truncations proved to be confined to a distinct region within the genome.

Capsid↗
PubMed1987

Replicative events in hepatitis A virus-infected MRC-5 cells.

Replication of hepatitis A virus (HAV) in MRC-5 cells was studied under one-step growth conditions. Viral replication neither interfered detectably with cellular DNA, RNA, and protein synthesis, nor could cytopathologic changes be recorded over a prolonged period of incubation. Synthesis of mature, infectious HAV particles could be detected as early as 2-4 days p.i. and occurred at a maximal rate around 8 days p.i., shortly before infectivity titers reached a plateau. Synthesis of total viral RNA, of positive-strand genomic RNA, of viral mRNA, as well as of negative-strand RNA followed the same pattern. By Day 14 p.i., when active HAV replication had developed into persistent infection, synthesis of viral RNA declined to background levels. The mechanism(s) guiding active HAV replication into a state of persistent infection could not be positively defined. Yet there exists the possibility that this is brought about by down regulation of viral RNA synthesis. Whether this is related to the appearance of a subgenomic viral RNA molecule about 2000 nucleotides in length and detected in association with ribosomes on Days 7 and 10 p.i. remains to be shown.

Capsid↗
PubMed1985

Variation among hepatitis A virus strains. I. Genomic variation detected by T1 oligonucleotide mapping.

The genomes of eight hepatitis A virus (HAV) strains originating from far distant geographic regions such as Europe, North Africa, Middle and North America, Australia and The People's Republic of China were compared by RNase T1 oligonucleotide mapping. For this purpose, the viruses were propagated in cell cultures and viral RNA was isolated from highly purified mature virions. It could be shown that variation in nucleotide sequence is common among HAV isolates, but is in the order of magnitude reported for other picornaviruses. For viruses isolated in cell culture directly from stool samples of diseased individuals, changes usually amounted to 1-4% of RNA genome sites. Genomic differences between two virus strains derived from one fecal sample but replicating at either 32 or 37 degrees C were in the same order of magnitude. Thereby, the number of consecutive in vitro passages proved to have only limited influence on the development of genetic variation. For two HAV strains, however, adaptation to and passage in marmosets evidently had imposed highly selective conditions which had favored the appearance of viral genomes differing in up to 75% of their large oligonucleotides (about 10% in sequence) from the oligonucleotide map of a reference HAV strain.

Animals↗
PubMed1984

Early events in the replication of parvovirus LuIII.

Purified infectious LuIII virions characterized for their biological and physicochemical properties were used to analyse early events in the interaction of an autonomous parvovirus with its host cell. It could be shown that adsorption and penetration of LuIII virus are rapid processes which, under optimal conditions, can be completed within 30 to 60 minutes of incubation at 37 degrees C. Uncoating of the viral genome is initiated in the cytoplasm and is continued after the virus has been transported to the nucleus. None of these processes appeared to depend on cell cycle-specific helper function(s). However, the first step in viral replication--formation of viral parental RF-DNA consisting of covalently linked complementary strands--evidently could only be achieved in late S-phase of the cell cycle. No evidence was obtained which would support the hypothesis that viral DNA must be integrated into the cellular genome to allow for the initiation of viral DNA synthesis.

Biological Transport↗
PubMed1984

Propagation and assay of hepatitis A virus in vitro.

Ten strains of hepatitis A virus (HAV) originating from far distant geographical locations were adapted to growth in PLC/PRF/5 (human hepatoma derived and/or MRC-5 (human embryonic lung) cells. In the course of primary adaptation some of these strains exhibited a predilection for distinct cultural conditions such as type of host cell and temperature of incubation. With progressive passage, variant viruses with quite different requirements could be selected; yet, it proved impossible to isolate a virus which replicated equally well in both types of cells and at both 32 and 37 degrees C without at least one preceding passage under the new conditions. Analysis of the virus/cell relationship of well adapted HAV strains revealed that the replication cycle of HAV extends over about 24 h. Moreover, replication evidently passes from a state of active production of infectious virus to a phase during which hepatitis A antigen (HAAg) is synthesized and terminates in the state of persistent infection with markedly reduced synthetic activity. In all three phases replication of HAV is non-cytolytic and the vast majority of both infectious virus and of HAAg remains cell associated. The observations concerning the growth characteristics of HAV were used to develop two rapid in vitro assay systems for HAV infectivity (fluorescent focus assay and in situ RIA). Finally, the conditions for large scale production of infectious HAV and of HAAg in a cell factory system were analysed.

Adaptation, Physiological↗
PubMed1984

Stability of hepatitis A virus.

The stabilities of hepatitis A virus (HAV) and of poliovirus type 2 were compared under strictly controlled, identical conditions of pH value, temperature, and salt concentration. Although the resistance of the viruses proved to be the same from pH 3 to 11, the temperature at which 50% of poliovirus particles became disintegrated during heating at pH 7.0 for 10 min (T50,10 = 43 degrees) differed significantly from that characteristic for HAV (T50,10 = 61 degrees). In the presence of 1 M MgCl2, the T50,10 for poliovirus and for HAV shifted to 61 degrees and 81 degrees, respectively. Destabilization of the physical integrity of HAV by heating resulted in the release of viral RNA and, simultaneously, in the generation of several 'empty' capsid structures or dissociation products thereof. Empty capsids and further dissociation products were still serologically reactive with anti-HAV IgG contained in human convalescent sera. With further heating (greater than T50,10 = 67 degrees or greater than T50,1 = 87 degrees), however, they irreversibly lost this reactivity.

Capsid↗
PubMed1983

Maturation of parvovirus LuIII in a subcellular system. I. Optimal conditions for in vitro synthesis and encapsidation of viral DNA.

The development of an in vitro system prepared by lysis of LuIII virus-infected cells with Brij-58 has enabled the study of the assembly pathway of a parvovirus. Under optimal conditions, radioactive precursors are incorporated both into viral replicative form double-stranded DNA and into progeny viral DNA (vDNA) during pulses as short as 30 s. Labelled 110S particles can be isolated at the end of such pulses. Therefore, synthesis and encapsidation of progeny viral DNA into pre-existing empty viral capsids appear to be closely related processes. Up to about 10 min after incorporation of vDNA, the 110S particles band at 1.44 g/ml and are relatively unstable in 3.5 M-CsCl. Moreover, newly synthesized vDNA molecules show an abnormal electrophoretic behaviour probably due to the presence of a covalently linked terminal protein. This so far uncharacterized alkali-stable polypeptide is lost (cleaved off?) concomitant with the maturation of the 110S virus particles. Maturation is reflected by a change in the virus stability in CsCl and a shift in density from 1.44 to 1.41 g/ml around 10 to 15 min after encapsidation of progeny vDNA.

Capsid↗
PubMed1983

Maturation of parvovirus LuIII in a subcellular system. II. Isolation and characterization of nucleoprotein intermediates.

Nucleoprotein (NP) intermediates in the assembly pathway of parvovirus LuIII were isolated. These structures consist of replicating viral DNA and of preformed viral capsids into which progeny viral DNA is encapsidated concomitant with synthesis. The NP complexes sediment at between 70S and 100S. They are unstable in CsCl and dissociate in the presence of 1 M-NaCl. After fixation with glutaraldehyde, however, they accumulate at a density of 1.37 g/ml in CsCl gradients. Electron microscopy of fixed complexes revealed structures consisting of DNA threads associated with capsid-like particles. A considerable proportion of the DNA labelled during short pulses in such complexes, in a subcellular in vitro system derived from infected nuclei, can be effectively chased into 110S virions. The resulting 110S particles apparently have to undergo a sequence of morphogenic events to acquire the physicochemical properties of the mature infectious parvovirus.

Cells, Cultured↗
PubMed1983

Taxonomic classification of hepatitis A virus.

Sufficient data have accumulated to permit the ICTV Ad Hoc Study Group on the Taxonomy of Hepatitis Viruses to recognize hepatitis A virus as a picornavirus. Within the family Picornaviridae, hepatitis A virus closely resembles members of the genus Enterovirus.

Enterovirus↗
PubMed1982

Failure of guanidine and 2-(alpha-hydroxybenzyl)benzimidazole to inhibit replication of hepatitis A virus in vitro.

Replication of hepatitis A virus (HAV) in the human hepatoma-derived PLC/PRF/5 cell line was neither inhibited in the presence of various concentrations of guanidine or D-2-(alpha-hydroxybenzyl)benzimidazole (D-HBB), nor were the two chemicals effective in combination. Under identical conditions, however, replication of poliovirus type 1 was inhibited. Tracer experiments with radiolabelled guanidine and D-HBB also furnished no evidence that the two antiviral substances were metabolized gradually to inactive derivatives in PLC/PRF/5 cells. Therefore, it is concluded that resistance to the action of guanidine and D-HBB is an inherent characteristic of HAV. However, the insensitivity of HAV to these drugs does not exclude the virus from the family of picornaviruses.

Antigens, Viral↗
PubMed1982

Canine parvovirus: relationship to wild-type and vaccine strains of feline panleukopenia virus and mink enteritis virus.

Canine parvovirus (CPV), feline panleukopenia virus (FPLV) and mink enteritis virus (MEV) were compared serologically, by determination of their host range in cell cultures, as well as by restriction enzyme analysis. Maps of the virus genomes were established using seven different restriction enzymes cutting at a total of 56 sites. MEV and FPLV gave maps which were identical except for one restriction site. The map of CPV is closely related to those of FPLV/MEV since their DNAs share about 80% of the restriction sites tested. However, CPV is clearly distinct from FPLV/MEV since either eight (German isolate) or nine (Belgian, Swiss and American isolates) restriction sites are different. The DNAs of six vaccine strains of FPLV and MEV were also analysed. They gave maps which closely resembled those of the respective wild-type strains. CPV and FPLV/MEV also differed with respect to antigenicity, as well as to host range in cell cultures.

Animals↗