Biphasic production of immune interferon during mitogen-induced blastogenesis of human peripheral blood mononuclear leukocytes.

Peripheral blood mononuclear leukocytes of 14 healthy blood donors stimulated by concanavalin A were shown to exhibit a biphasic pattern of the production kinetics of interferon. Maximum interferon titers were determined in the culture supernatants 8 and 48 h after addition of the mitogen. Both the early and late interferon production could be abolished by actinomycin D treatment of the induced cells implying that they depend on mitogen-stimulated de novo synthesis of cellular RNA. Under the conditions used the interferon mRNA was accumulated in treated cells during the first 4 h of incubation. The kinetics as well as the efficiency of concanavalin A-induced interferon production turned out to strictly depend on the nature of the protein source added to the culture medium. Only fetal calf serum and calf serum as compared to human albumin or pooled gamma globulin-free human plasma were found to give the highest interferon yields in true biphasic production pattern, thus explaining, at least in part, the differing data obtained by several investigators working with this particular interferon production system.

[Improved test for the detection of reverse transcriptase of bovine leukosis virus]. / Ein verbesserter Test zum Nachweis der reversen Transkriptase des Rinderleukosevirus.

The revertase test with exogenous matrix, poly-(rA) . oligo-(dT), for the detection of bovine leukemia virus from crude virus sediments was standardised and miniaturised. An amount of 10 ml of cell culture supernatant of short-term cultured lymphocytes (5 . 10(6) cells/ml) is quite sufficient for testing one cattle sample. The lower sensitivity limit of the test was found to be 10(6) virus particles. The test is properly reproducible, within tolerance limits of +/- 20--30 per cent, provided that optimum lysis conditions be maintained (0.01 per cent triton X-100, 20 minutes, 0--4 degrees C incubation) and under the condition that the protein quantity in 100 microliter test solution does not exceed the threshold of 3--15 micrograms. The specificity of the test is based on the use of free viruses from cell culture supernatant, the optimum temperature of the revertase reaction at 25 degrees C, which actually deviates from that for cellular DNA polymerases, that is 37 degrees C, and magnesium ion concentration which has to be optimum for bovine leucosis virus revertase. Two-hundred heads of cattle, differing by haematological status, were examined, and 56 per cent of them were, clearly, virus producers, among them 95 per cent of all animals with positively established leucosis and 36 per cent of the haematologically intact animals. Examinations of individuals have shown that in repetitive checks, carried out in intervals between two months and one week, the revertase activities varied by something between 0.5 and two magnitudes.

Purification and characterization of bovine leukemia virus DNA polymerase.

An RNA-directed DNA polymerase was purified from bovine leukemia virus (BLV) by successive glycerol gradient centrifugation, column chromatography on phosphocellulose and gel filtration on Sephadex G-200. The purified DNA polymerase transcribes heteropolymeric regions of 30--40 S RNA isolated from avian myeloblastosis virus. The enzyme differs from other known DNA polymerases of mammalian type-C RNA tumor viruses by the following properties: 1. Its apparent molecular weight as estimated by velocity sedimentation data is 58,000 at 0.12 M KCl and 43,000 in the presence of 0.50 M KCl. 2. It has a Mg2+ optimum of 10 mM, and a Mn2+ optimum of 0.25 mM with (rA)n-(dT)10 as template. 3. At 50 mM KCl it is inhibited more than 70%, but it is not inhibited by phosphate ions at 2 mM. These properties confirm the peculiar position of BLV within the family Retraviridae.

[Studies on the virus demonstration in enzootic bovine leukosis using the revertase test]. / Untersuchungen zum Nachweis des Virus der enzootischen Rinderleukose mit Hilfe eines Revertasetestes.

This exogenic test for the detection of enzyme activities (revertase) is helpful in differentiating between leucocytes of leukaemic cattle, on the one hand, and those of intact animals, on the other. The mechanism of such identification is based on differences in enzyme activity in the presence of poly rA/oligo dT and poly dA/oligo dT. The best results were obtained from short-time cultured leucocytes which gave ten times higher margins of incorporation by comparison between intact and preleucotic cattle. The morphological findings obtained by electron microscopy confirmed the results by other authors and enlarged them by determination, for the first time, of RNA filaments and protein rings.

[Structures of oncornaviruses of C-typ-negativ staining technique by electron microscopy (author's transl)]. / Strukturen der Onkornaviren vom C-Typ--Elektronenmikroskopische Untersuchungen im Negativkontrastverfahren.

Typ-C viruses of bovine enzootic leucosis and other typ-C viruses were demonstrated by negativ staining technique and also the RNA-protein complex respectively the nucleocapsid of helical symmetry in the shape of coral bead. Now the well defined morphology of the typ-C viruses enable to identificate to be used in electron microscopy practice.