Propagation of hepatitis A virus in human embryo fibroblasts

human diploid fibroblasts and human primary liver cell carcinoma cells (PLC/PRF/5) were infected with hepatitis A virus (HAV) adapted to growth in cell culture or derived directly from human stool. Viral antigen was expressed in PLC/PRF/5 cells 28 days after infection with cell culture-adapted HAV, and 50 days after infection with virus from human stool. In human fibroblasts the periods until first expression of viral antigen were 90 and 210 days, respectively. During further passages of HAV in fibroblasts the time of first appearance of antigen decreased to about 28 days. Biophysical properties of HAV extracted from infected fibroblasts were comparable to those of HAV derived directly from human stool. Immunofluorescence studies showed that the antigen was located exclusively within the cytoplasm of the infected fibroblasts. Kinetics of antigen production indicated that an equilibrium between virus multiplication and cell metabolism was reached in persistently infected fibroblasts.     

Expression of human immunodeficiency virus type 1gag gene using genetically engineered herpes simplex virus type 1 recombinants

Infectious herpes simplex virus type 1 (HSV-1) recombinants were constructed by inserting the cDNA sequence of the human immunodeficiency virus type 1 (HIV-1)gag gene (from nucleotide position 675 [SacI] to 3859 [Asp 718] of the cDNA sequences of HIV-1 strain BH-10) within the DNA sequences of theBamHI DNA fragment B of the genome of an apathogenic HSV-1 strain HFEM. This HSV-1 strain possesses a 4.1-kbp deletion within theBamHI DNA fragment B between 0.762 and 0.789 map units of the viral genome, which allows the insertion of at least 4 kbp of foreign genetic material into this particular region. The DNA sequences of the immediate early promoter (IE4) of HSV-1 that were inserted upstream from thegag gene were used as a promoter. The screening of 205 virus stocks derived from individual plaques revealed that 46 recombinant viruses harbor HIV-1gag-specific DNA sequences. However, it was found that only six of the recombinant viruses are able to express thegag gene product of HIV-1. This indicates that the ratio of the positive recombination events is about 2.9%.     

Association of human leucocyte low responsiveness to inducers of interferon alpha with HLA-DR 2

The influence of antigens of the major histocompatibility complex (MHC) on the production of interferon (IFN) alpha or IFN gamma by human peripheral blood leucocytes (PBL) in vitro has been studied. Synthesis of IFN gamma by PBL stimulated with purified phytohaemagglutinin (PHA-P) or protein A of Staphylococcus aureus (SpA) appeared not to be controlled by MHC antigens. The production of IFN alpha, however, was influenced by the HLA type of the donor. Low responsiveness of PBL to inducers of IFN alpha (influenza virus, Molt 4 cells) was associated with HLA-DR 2. Implications of these observations for studies of IFN production and natural killer (NK) cell activity are discussed.     

Prevalence of human T-Cell lymphotropie virus infections in Germany

The extent of human T-cell lymphotropic retorvirus HTLV-I and HTLV-II infections in the general population in central Europe has not been investigated fully. Two hundred forty-eight thousand blood donors from southern Germany were examined serologically for antibodies to the human lymphotropic retroviruses HTLV-I and HTLV-II: 0.021% were confirmed postive and 0.056% were “indeterminate”. A limited number of seropositives and “indeterminate” samples were analyzed by polymerase chain reaction (PCR): the seropositives were confirmed as positive and 43% of the “indeterminate” samples were PCR-positive. The range of 0.021% HTLV-positives in 248,000 donors, i.e. about two in 10,000 individuals, mirrors closely the published data for the United States. © 1994 Wiley-Liss, Inc.     

Immunoglobulin M antibodies to hepatitis B core antigen: evaluation of enzyme immunoassay for diagnosis of hepatitis B virus infection

In acute and chronic hepatitis B, antibodies of the immunoglobulin M (IgM) class against the hepatitis B core antigen (anti-HBc IgM) have been demonstrated. For the determination of anti-HBc IgM, a sensitive enzyme immunoassay with anti-mu-coated flat-bottomed microtiter plates is described and evaluated. The specificity of the anti-HBc IgM test system was proven by pretreatment of presumed anti-HBc IgM-positive samples with anti-mu to block anti-HBc IgM. The test system was highly sensitive. In the acute stage of hepatitis B, anti-HBc IgM could be demonstrated in serum dilutions up to 10(-7) (mean titer, 10(-5)), and in sera from patients with chronic hepatitis B, the mean titer was 10(-3). In a study of unselected patients whose sera were sent at irregular intervals for testing, anti-HBc IgM persisted in a high percentage (52%) for at least 13 to 18 months after onset of illness despite the fact that these patients eliminated hepatitis B surface antigen (HBsAg) and produced antibodies to HBsAg (anti-HBs). By using the anti-HBc IgM test as an additional aid in the diagnosis of acute HBsAg-negative hepatitis, the hepatitis B etiology could be established in 13 of 42 patients (31.4%). Investigations of the prevalence of anti-HBc IgM in different groups of patients with chronic hepatitis B infection showed 89.4% anti-HBc IgM-positive results in patients with chronic active hepatitis B, 60% in patients with HBsAg-negative chronic active hepatitis, 58.2% in patients with primary liver carcinoma and markers of hepatitis B infections, and 34.9% in healthy carriers of HBsAg.     

A new second-generation anti-HIV-1 enzyme immunoassay using recombinant envelope and core proteins

In a multicenter collaborative study a new second-generation HIV-1 antibody enzyme immunoassay (Abbott recombinant HIV-1 EIA) using Escherichia coli-expressed recombinant p24 and p41 proteins as solid-phase antigens was compared with the first-generation H9 cell-line-based Abbott HIV-1 EIA. The results of the confirmatory assays (Western blot, immunofluorescence), combined with clinical information, were used as the reference standard for the detection of HIV-1 antibodies in 10,676 random blood donor serum specimens, in a panel of 840 specimens from symptomatic and asymptomatic patients and a total of 63 serial blood specimens from 23 people at risk. With fresh blood donor sera, the specificity of the first-generation assay ranged between 99.54 and 99.76% (95% confidence limits, CL) compared with 99.81-99.95% (95% CL) for the second-generation EIA. With panel specimens the recombinant HIV-1 EIA achieved an overall sensitivity of 100% and a specificity range of 98.3-99.7% (95% CL); the corresponding sensitivity and specificity ranges observed for the first-generation EIA were 98.0-99.5% (95% CL) and 94.3-96.8% (95% CL), respectively. The improved sensitivity for the second-generation assay was confirmed by testing serial samples from seroconverting patients. The use of recombinant proteins eliminated non-specific reactions due to class II human leukocyte antigen (HLA)-directed antibodies.     

Interferon production in male homosexuals with the acquired immune deficiency syndrome (AIDS) or generalized lymphadenopathy

Summary We measured serum interferon (IFN) levels and leukocyte IFN productionin vitro in homosexual men with AIDS or generalized lymphadenopathy and in asymptomatic homosexual or heterosexual men. Acid-labile interferon alpha was detected in the serum of two patients with AIDS and in three out of nine patients with lymphadenopathy, but not in the sera of healthy homosexual or heterosexual controls. The presence of serum interferon in patients with AIDS or lymphadenopathy was correlated with a marked deficiency of their leukocytes to synthesize interferon alpha in response to viruses or tumor cells, but did not significantly interfere with the production of interferon gamma in response to lectin or bacterial enterotoxin. Our results suggest an inverse correlation between serum interferon alpha and the amount of interferon alpha produced in leukocyte cultures in male homosexuals with AIDS or generalized lymphadenopathy.

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Bildung von Interferon bei Patienten mit AIDS

Zusammenfassung Wir haben Messungen der Serum-Interferon-Spiegel und der Interferon-Bildung durch Leukozytenin vitro bei homosexuellen Männern mit AIDS oder generalisierter Lymphadenopathie und bei gesunden homosexuellen oder heterosexuellen Männern durchgeführt. Säurelabiles Interferon alpha wurde im Serum von zwei Patienten mit AIDS und bei drei von neun Patienten mit Lymphadenopathie, aber nicht im Serum gesunder homosexueller oder heterosexueller Kontrollpersonen gefunden. Das Vorhandensein von Serum-Interferon bei Patienten mit AIDS oder Lymphadenopathie korrelierte mit einer deutlichen Verminderung der Interferon-alpha-Synthese ihrer Leukozyten nach Induktion mit Virus oder Tumorzellen, beeinflußte jedoch nicht signifikant die Bildung von Interferon gamma nach Induktion mit Lektin oder bakteriellem Enterotoxin. Unsere Ergebnisse lassen eine inverse Korrelation zwischen Serum-Interferon alpha und der Menge des in Leukozytenkulturen gebildeten Interferon alpha bei männlichen Homosexuellen mit AIDS oder generalisierter Lymphadenopathie vermuten.

     

Productive infection of in vitro generated haemopoietic progenitor cells from normal human adult peripheral blood with parvovirus B19: studies by morphology, immunocytochemistry, flow-cytometry and DNA-hybridization

Parvovirus B19 exerts a highly selective cytopathic effect on erythroid progenitor cells. Studies so far on the pathogenesis of B19-infection have been performed using bone marrow samples providing large amounts of erythroid progenitor cells. Extensive study, however, has been hampered by the limited access to bone marrow samples. We have designed a liquid culture method allowing the generation of large numbers of erythroid progenitor cells, initiating cultures with CD3- and CD14-poor peripheral blood mononuclear cells. Following a 12 d preincubation in liquid cultures containing recombinant human interleukin 3 (rhIl-3) and recombinant human erythropoietin (rhEpo), cells harvested from the liquid cultures were exposed to B19-containing plasma, followed by a further cultivation in liquid culture for up to 96 h. Cells expressing the CD13 and the glycophorin A (GlyA) antigens, respectively, were monitored sequentially by flow-cytometry, demonstrating a selective inhibition of GlyA-positive cells following B19-inoculation. Typical morphological changes were observed on cytocentrifuge-spots, and typical giant-cells were identified as staining for GlyA. Productive infection by B19 was demonstrable, as B19-DNA increased by about x 100 after 72 h of culture. The liquid culture method generating erythroid target cells for effective infection by B19 virus promises to be a useful and easily accessible tool for further research on B19 infection of haemopoietic cells.