Interferon assays are based on the inhibition of either viral function or synthesis of virus or its components. No one assay has proven to be ideal but significant improvements are constantly being introduced ( 1 ) . A study was initiated to develop a widely applicable assay which would combine rapidity, simplicity and reliability and which is based on the inhibition of virus multiplication during a single cycle of growth.Materials and Methods. Cell cdtures. Mouse L cells (obtained from Dr. J. S. Youngner, University of Pittsburgh) primary and secondary mouse embryo cells (NIH Swiss), primary chicken embryo cells and 2 diploid human cell lines (MA-308 and RCTC-2) were cultured to confluency by standard procedures. For assay, cells were cultured in 16 x 150 mm tubes, held vertically in a spring rack ( 2 ) and covered loosely with a single metal lid (3 ) .Viruses. GD-7 virus, a mouse picornavirus, was produced and plaque-titrated [ 106.9 plaque-forming units (pfu)/ml] in BHK-2 1 cells as previously described (4). Sindbis virus was prepared and plaqued pfu/ml) in chicken embryo monolayer cultures. Vesicular stomatitis virus (VSV) , Indiana strain, was also prepared in chicken embryo cells but the plaque titration was done in mouse L cells ( 107v5 pfu/ml).Interferons. Mouse serum interferon (MSI) ( 5 ) titered 104s2 units/ml in mouse L cells. Human interferon was prepared from human diploid fibroblast cell cultures infected with Chikungunya virus [ input multiplicity of infection (MOI) = 11 and it titered 103.0 units/ml in MA-308 cells. Chicken interferon induced in ovo with NWS influenza virus (6) titered 102a3 units/ml in chicken embryo cells. These titers were obtained using the methods described below.Hemagglutination assay. Titration of GD-7 virus hemagglutinin (HA) was done by standard methods as previously reported (4). 0.06% fetal bovine serum (FBS) was added both to erythrocyte suspensions and to the virus diluent because it was found to be essential for the proper development of the hemagglutination patterns. Titration of Sindbis virus HA was done as previously reported (7) using the determined optimum pH of 6.0 and a 0.125% concentration of gander erythrocytes in the final reaction mixture.
Results. Time course of H A production.To determine the times of maximal HA production in the virus-cell systems under study, single step growth curves were determined (Fig. 1 ) . It may be seen that maximum HA production occurred at 1 2 hr for Sindbis virus in chicken and mouse embryo cells, at 16 hr for Sindbis virus in human MA-308 cells and 18 hr for GD-7 virus in mouse L cells.The time course of viral production in interferon treated cells is generally not different or only slightly delayed (8). Therefore, during interferon assays lfluids were harvested for HA determinations 18-24 hr after infection.Interferon assay b y inhibition of yield of viral H A . Duplicate or triplicate tube cultures of cells were each exposed to l ml of interferon diluted in Eagle's minimal essential medium (EMEM) plus 2% FBS. After 18-24 ...
The single-stranded synthetic RNAs, polycytidylic (poly-rC) and polyinosinic (poly-rI) acids, can induce interferon-like resistance in various cell species (1). However, individual preparations varied widely in their activity. Possible explanations for this variation include differences in absorption by cells and destruction by cellular ribonucleaseIn order to test this hypothesis we investigated the possibility that substances which are known to improve cellular uptake of RNA, or to inhibit RNase activity, would enhance the resistance-inducing effect of single-stranded poly-rI, poly-rC, and poly-rI: rC. Specifically we tested methylated albumin, protamine sulfate, and DEAE-dextran which were reported to both enhance uptake (2)(3)(4)(5)(6)(7) and to inhibit degradation by ribonuclease (3, 7). Neomycin and streptomycin which inhibit ribonuclease activity ( 8) were also tested.
Materials and Methods. Compounds. Sin-gle-stranded polyinosinic acid ( poly-rI ) and polycytidylic acid ( poly-rC) were purchased from three different manufacturers: (i) P-L Biochemicals, Inc., Milwaukee, Wisconsin, (ii) Miles Chemical Co., Elkhart, Indiana, and (iii) Biopolymers, Chagrin Falls, Ohio. The compounds were dissolved in phosphate buffered saline containing 100 units/ml of penicillin and 100 pg/ml of streptomycin. Stock solutions containing 1000 pg/ml were kept frozen a t -20". Double-stranded poly-r1:rC was prepared by mixing equal volumes of stock solutions of poly-rI and poly-rC and (1).
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