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The attenuation of two human influenza A viruses has been carried out, using the selection of inhibitor-resistant strains and multiple passages at low temperatures. A virus related to A2/Tokyo/3/67 was obtained in an inhibitor-resistant form. When this was compared with the inhibitor-sensitive strain in a volunteer trial it was relatively non-pathogenic. The second virus, A2/Hongkong/1/68, was subjected to much longer treatment, but nevertheless remained slightly sensitive to serum inhibitor. When given to volunteers it was less pathogenic than before but attenuation was incomplete. A2/Hongkong/1/68 was also modified by passage at low temperatures. Many of these passages are apparently necessary for full attenuation. All attenuated viruses were infective and antigenic.

The acetone-soluble fraction extracted from lyophilized cells of Mycobacterium smegmatis ATCC 607 inhibited D4, a species-specific phage active against M. smegmatis . Evidence is presented indicating that the D4 inhibition was caused by the phage receptor substance(s) contained in this fraction. A fraction eluted from silicic acid with chloroform-methanol (95:5, v/v) showed the strongest inhibition of D4 phage. This fraction contained sugars and amino acids, and its infrared absorption spectrum was practically identical with those of the mycoside C isolated from the other species of mycobacteria. Further fractionation revealed that the active material was a mixture of several closely related peptidoglycolipids all of which showed, more or less, the phage inhibition. One of the compounds was purified and partially characterized; it contains three different amino acids, allo -threonine, alanine, and phenylalanine, at a molar ratio of 1:1:1, and also three different deoxyhexoses, probably 6-deoxytalose, 3,4-di- o -methylrhamnose, and 2,3,4-tri- o -methylrhamnose. A tentative name of “mycoside C sm ” is proposed for this substance which possesses a slightly different structure from the known types of mycoside C and is probably specific for the species of M. smegmatis . A fraction extracted from the D4-resistant mutant of M. smegmatis ATCC 607 by acetone and then by chloroform-methanol (95:5, v/v) showed no phage inhibition and had no sugar component. In addition, this fraction contained lysine, serine, and a small amount of both glycine and an unidentified amino acid.

Streptococcus pyogenes survives poorly on plain cotton-wool swabs, whereas serum-dipped swabs permit its survival but also allow overgrouth by other bacteria and are likely to contain virus inhibitors. The use of swabs dipped in a solution of 20% bovine albumin (fraction V) and sterilized by steam is therefore recommended, since these permit bacterial survival without growth and are unlikely to contain virus inhibitors.

The isolation of antitumor active oily substances from the mycelia of Penicillium crustosum, Penicillium tardum, Cephalosporium diospyri and Sepedonium ampullosprum has been described previously. The present paper describes chemical studies on these active products. Their infrared absorption spectra were virtually identical and were typical of long-chain fatty acids.Nuclear magnetic resonance spectra confirmed the presence of long-chain methylene and methylene interrupted double bonds. Mass spectra and the gas chromatographic studies of the methyl esters indicated the presence of palmitic, octadecaenoic and octadecadienoic acids as major constituents.

Early in the investigations on interferon, Isaacs and Lindenmann obtained suggestive evidence that the nucleic acid of the virus was responsible for the stimu- lation of interferon production in cells. Recently, Rotem, Cox, and Isaacs2 reported that RNA3 of animal source, heterologous to the tissue in which it was tested, was also able to produce interferon. This observation provided an explana- tion for the results of O'Dell, Wright, and Bieter,4 who as early as 1953 showed that yeast RNA inhibited MM virus in mice. In our laboratory we have continued the investigation of the properties of stato- lon, an antiviral agent produced by the mold Penicillium stoloniferum.5 It has proved to be prophylactically active against a wide spectrum of viruses in tissue culture and in animals.6-"3 Statolon is a complex anionic polysaccharide with a relatively high content of galacturonic acid, which is the primary contributor to its Early in the investigations on interferon, Isaacs and Lindenmann obtained suggestive evidence that the nucleic acid of the virus was responsible for the stimu- lation of interferon production in cells. Recently, Rotem, Cox, and Isaacs2 reported that RNA3 of animal source, heterologous to the tissue in which it was tested, was also able to produce interferon. This observation provided an explana- tion for the results of O'Dell, Wright, and Bieter,4 who as early as 1953 showed that yeast RNA inhibited MM virus in mice. In our laboratory we have continued the investigation of the properties of stato- lon, an antiviral agent produced by the mold Penicillium stoloniferum.5 It has proved to be prophylactically active against a wide spectrum of viruses in tissue culture and in animals.6-"3 Statolon is a complex anionic polysaccharide with a relatively high content of galacturonic acid, which is the primary contributor to its polyanionic character.5 Structurally, RNA also is a polyanion. The finding by Rotem et al.2 that heterol- ogous RNA stimulated the production of interferon suggested that the mechanism of action of statolon possibly lay in its capacity to stimulate the production of interferon. This possibility has been investigated, and the results presented in this paper show that statolon induces the production of a viral inhibitor in cells, and that this inhibitor has the properties of interferon. Materials and Methods.-Statolon: A freeze-dried preparation was used that contained 89.47% sodium bicarbonate and glucose, in equal proportion."4 The statolon was dissolved in water, 50 mg/ml, and the pH adjusted to 7.5 with CO2. The solution of statolon was diluted 1:300 with medium 199, bringing the concentration of active statolon to 17.5 tAg/ml. Virus: Vaccinia virus, strain V-l, was obtained through the kindness of Dr. J. Lindenmann, University of Florida. Stock virus was prepared in HeLa cells and was stored in sealed glass ampoules at -60?C. Cells: Primary cultures of chick embryo (CE) cells from 1-day-old embryos were employed. Production of induced inhibitor: 70 X 106 CE cells in 30 ml of medium 199 containing 4% calf serum were added to rectangular pint bottles with a surface area of 100 cm2 and incubated 48 hr. The medium was then replaced with 30 ml of a statolon solution (17.5 ,ug/ml), the cells were incubated another 24 hr, and the fluid was harvested. Residual statolon was removed from the fluid by concentrating it fivefold in dialysis bags by means of polyethylene glycol, then lowering the pH to 4.5 and centrifuging for 90 min at 105,000 X g in the Spinco (Model L) centrifuge. The supernatant fluid was removed from the tubes and the pH was adjusted to 7.3. Assays showed that statolon solutions that had never been in contact with cells no longer possessed inhibitory activity after such treatment. After removal of the statolon, fluids were diluted 1:5 to the initial

THE ability of the intestinal content and intestine of normal mice to inactivate murine hepatitis virus (MHV-3) was recently reported1. The present paper deals with the localization of this activity in different sections of the intestine and in separate strata of the intestinal wall. The distribution of the inhibitor has also been studied in mice of varying ages, and in various tissues of mice, as well as in the intestine of several other species. The interaction between the inhibitor and the virus has also been studied in vitro at 37° C and at 2° C.

ETHIDIUM bromide (EB) (2,7 di-amino-phenyl phenanthridine 10-ethyl bromhydrate) is an antimicrobial drug which, like proflavine (an acridine), forms reversible complexes with nucleic acids1,2. In vitro, it inhibits DNA-polymerase and, at a lesser rate, DNA-dependent RNA-polymerase2. In the same way, EB and related phenanthridine derivatives inhibit the synthesis of nucleic acids in bacteria3 and Ehrlich ascites cells4; they are also trypanocidal5. Moreover, the photosensitization of arboviruses6, the inhibition of their development7 and that of a Pseudomonas pyocyanea phage8 by these compounds have been reported. Although the responses of several virus-infected systems to the action of acridines have been given in detail9,10, the comparable interactions with EB have not yet been so clearly described. The present paper details the action of EB on herpes virus and its growth in baboon kidney cells.