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A survey has been made of the activity of a wide variety of standard strains of streptococci against bovine submaxillary mucin. Strain 6646 (group K) and strain D 168A "X" (group M) completely broke down and strain H 60R (group F) incompletely broke down bound sialic acid of bovine submaxillary mucin added to the growth medium. Among these strains, strain 6646 (group K) produced sialidase in the cell and in the culture fluid. An appropriate amount of glucose in the culture medium stimulated growth and the production of enzyme, but an excess of glucose in the culture medium caused abundant growth without production of the enzyme. The streptococcal sialidase was precipitated from the culture fluid by ammonium sulfate at 50% saturation, and further purification was achieved by diethylaminoethyl cellulose chromatography. Ca(++) and Co(++) stimulated the sialidase activity, and Mn(++), Zn(++), and ethylenediaminetetraacetate inhibited it. With acetate buffer, the optimal pH lay between 5 and 6. Sialic acid was detected in the reaction product of the streptococcal sialidase and bovine submaxillary mucin.

Neuraminidase activity has been found in a variety of strains of Corynebacterium diphtheriae, both toxinogenic and nontoxinogenic. The enzyme has been shown to be intracellular, possibly associated with the cytoplasmic membrane. Toxinogenic strains of the diphtheria bacillus, grown under conditions unsuitable for maximal toxin production, produce neuraminidase, and the enzyme has been purified from cells of the Park Williams no. 8 strain grown under such conditions. Diphtherial toxin and diphtherial neuraminidase have similar molecular weights and remain associated during column chromatography; immunochemically, and in their electrophoretic behavior, they appear distinct.

Neuraminidase (Nase) activity of chick embryo monolayer cell homogenates was determined by its rate of splitting of neuraminlactose, free neuraminic acid (NA) being determined by the thiobarbituric acid assay. Noninfected cells were found to have no detectable amount of Nase activity. Newcastle disease virus (NDV)-infected cells (multiplicity of infection, 20 to 75 plaque-forming units per cell) displayed a high level of Nase synthesis, the rate of synthesis being parallel to that of hemagglutinin (HA) synthesis (with a 1.5 hr delay in the latter). An "eclipse" of the Nase and HA activities associated with the virus that was adsorbed onto cells was observed. The data provide evidence that the Nase is not incorporated into the viral envelope from a pre-existing cell supply but that its synthesis is coded by the viral genome. The content of cell-bound sialic acid, determined simultaneously in infected-cell homogenates, showed characteristic features allowing certain conclusions concerning the renewal of NA-terminating cell receptors during the course of infection, and the intracellular action of the Nase of the virus introduced into cells by the inoculum and that of the newly synthesized Nase at different stages of infection.

Specific antisera for hemagglutinin (HA) and neuraminidase antigens of influenza A(2) virus (A(2)E) were produced through the segregation of the two proteins in reciprocal viral recombinants of A(2)E and A(0)e viruses. Gamma globulin fractions of these specific antisera and of antiserum specific for the nucleoprotein (NP) antigen of A(0)e virus were conjugated with fluorescein isothiocyanate and employed to follow the synthesis of the three structural proteins in clone 1-5C-4 human aneuploid cells, with parallel measurement of serological and biological activity of the antigens by other techniques. In this system, NP antigen appeared first (at 3 hr) in the cell nucleus, whereas HA and neuraminidase appeared coincidentally, at 4 hr after infection, in the cytoplasm. The initial detectability of biological or complement-fixing activity of the proteins coincided with their demonstrability as stainable antigens. Late in infection, all three antigens were detected at the cell surface. Antibody specific for HA partially blocked the intracellular staining of neuraminidase and inhibited the enzymatic activity of both extracted and intact extracellular virus. These observations suggest the close intracytoplasmic proximity of the two envelope antigens and perhaps their initial association in a larger protein.