SUMMARY: A total of 978 cultures of heterotrophic organisms were isolated from twelve actively nitrifying soils ; each isolate was tested for ability to form nitrite or nitrate in glucose peptone broth. None of the isolates yielded substantial amounts of nitrite ; concentrations found did not exceed 2 pg. nitrite-N/ml. Almost 7 yo of the isolates formed nitrite-N in excess of 0.2 pg./ml., while slightly over 2 yo yielded more than 0.5pg./ml. Fungus isolates were the most numerous and most active nitrite producers ; fifteen of the fungi formed nitrate in addition to nitrite. Concentrations of 5-45 pg. nitrate-N/ml. were recorded for the active fungi. Further isolations of fungi from ten soils of diverse properties resulted in 353 cultures but only three of these formed nitrate. One of these cultures yielded 9Opg. nitrate-N/ml. after 14 days growth in the test medium.Most of the fungi which produced nitrate (16 of the 18 active cultures) were identified as Aspergillus jlavus. A Penicillium sp., and a Cephalosporium sp. also formed low concentrations of nitrate. Each isolate of A . flavus obtained from soil proved capable of nitrate formation. Strains of A . flavus were encountered which did not yield nitrate, but these had been obtained from culture collections, and had been carried on artificial media for many years. Some stock cultures of A . jlavus, several cultures of A . jlavus and one of A . glaucus, freshly isolated from wheat seed, also were active nitrate producers. Ammonium sulphate and urea media supported growth of soil fungus isolates but not nitrate formation. Other organic nitrogen substrates in order of increasing effectiveness in nitrate production were : yeast extract, peptone, Protone and casein.

Enzymatic markers and electron microscopy were utilized to determine the cellular origin of the membrane types isolated from type 2 dengue virus-infected BHK cells by discontinouous sucrose gradient centrifugation. The results showed an apparent separation of plasma membrane, smooth and rough endoplasmic reticulum with increasing density. Virus-induced protein and RNA synthesis, as indicated by the incorporation of radiolabeled precursors, was localized on the rough endoplasmic reticulum. Glycosylation, measured by the incorporation of radiolabeled glucosamine into membrane-associated proteins, was most active in the bands of intermediate and smooth endoplasmic reticulum. Polyacrylamide gel electrophoresis of isolated membrane bands, radiolabeled in the presence of actinomycin D, after pulse inhibition by cycloheximide, revealed seven virusspecified proteins associated with all membrane fractions. Viral structural protein V-3, and nonstructural proteins NV-3 and NV-2, increased with decreasing density, whereas NV-5 and NV-4 remained constant. The viral capsid protein V-2 was depleted in the intermediate and smooth endoplasmic reticulum, suggesting that these membranes may serve as the sites for viral maturation. NV-3 was the most prominent virus-specified protein found in the plasma membrane.

The membranes isolated from type 2 dengue virus-infected BHK-21/15 cells contain three glycosylated virus-specified proteins; one corresponds to the virion envelope glycoprotein, V-3, whereas the other two are nonstructural virusspecified proteins, NV-2 and NV-3. A combination of two nonionic detergents, Nonidet P-40 and Triton X-305, solubilized .80% of the membrane-bound protein and the majority of the type 2 dengue virus complement-fixing antigens. The soluble material was adsorbed by concanavalin A-Sepharose in the presence of the nonionic detergents, which were subsequently removed by washing with deoxycholate-containing buffer. Finally, the bound glycoprotein was eluted by the addition of a-methyl glucopyranoside. V-3 was the only virus-specified protein in the a-methyl glucopyranoside eluate. The V-3-containing fraction did not cross-react with antisera against other selected Flaviviruses in the complement fixation test. The V-3 contained in the isolated fraction differed from the parent membrane-bound V-3 in two interesting, and as yet unexplained, ways: (i) it exhibited hemagglutinating activity similar to that of the infectious virus, but (ii) it did not block the action of neutralizing antibody.