When certain compounds-mostly antimetabolites inhibitory to influenza virus replication in tissue culture-were combined in various ways, the virus inhibition was enhanced in a s nergistic manner with many o f the combinations studied. The selectivit of this ingibitory effect also was increased significantly in most cases. I n view of tiese experimental results, further work seems warranted o n aerosol inhalation treatment of influenza-infected mice with certain combinations of these compounds. NUMBER OF analogs of amino acids and a t A least one enzyme have been shown to inhibit the replication of one or more animal viruses in tissue culture. However, the concentrations required usually have been relatively high and the ratio between toxic and inhibitory concentrations rather low. During the course of a study of influenza virus inhibitors, it appeared worthwhile to determine whether either of these limitations could be influenced favorably by combining two or more inhibitory compounds. The results of experiments employing various combinations of such inhibitors are the subject of this report.
EXPERIMENTAL
Influenza Virus.-The Lee strain of virus was employed. It was maintained in the form of frozen chick embryo allantoic fluid stored a t -60" and was titrated periodically for infectivity in 10day chick embryos by the method previously described (1).
Compounds.-L-Canavanine was extracted from jack bean meal and isolated as the free base; lupulon was extracted from hops and purified. Both were prepared in the Department of Chemistry, Oregon State University, through the kind cooperation of Dr. B. E. Christensen. All of the other experimental compounds were obtained from the Nutritional Biochemicals Corp., Cleveland, Ohio.
Tissue Culture Method.-The tissue culture system employed also has been described (1). About 4 cm.2 of chorioallantoic tissue from 10-day chick embryos was suspended in 2.0 ml. of Hanks' balanced saline solution (BSS) in 25 X 150 mm. Pyrex culture tubes. Compounds to be studied were dissolved in this solution and the pH adjusted to 7.5 by addition of 1.4y0 NaHCOa solution or 0.03 N HCl. Such solutions were sterilized by filtration through fritted glass filters. Penicillin was incorporated in a concentration of 10 units/ml. and streptomycin at 40 mcg./ml. Cultures were