I-Adamantanamine (amantadine) causes a selective, reproducible, dose-related inhibition of influenza infections in tissue culture, chick embryos, and mice. The compound is not virucidal and appears to act by interfering with the penetration of the host cell by the virus. In influenza infections of mice, greatest efficacy occurs with treatment at the time of infection; however, there is significant antiviral activity with treatment delayed up to 72 hours after infection. Virus inhibition is not complete and survivors are immune to a challenge infection with the original infecting virus.

The antiviral activity of l-adamantanamine hydrochloride+ (amantadine HCI) in tissue culture, in ovu and in vivo has been briefly reviewed by Davies et aZ( 1) and Maassab and Cochran( 2 ) . Demonstration of corresponding antiviral activity in human studies has been presented by Jackson et aZ(3) and Wendel(4). In this paper is presented a detailed account of the antiviral activity of amantadine HCl in tissue culture and in ovo.Materials and methods. Compound. Solutions of amantadine were made either from the free base (neutralized with HCI) or from the hydrochloride salt.Media. All media contained 100 units of penicillin G and 100 p g of streptomycin sulfate. The basic maintenance medium consisted of 90% Earle's saline(5) modified to contain 0.9% NaCl, 0.45% glucose, 0.015% phenol red and 0.5 % enzymatic lactalbumin hydrolysate (Nutritional Biochemicals) and 10% Difco tryptose phosphate broth with 0.1% yeast extract and 290 mg% glutamine added. For studies on plaque formation 0.9% agar and for hemadsorption studieb 0.3% agar was added to the liquid maintenance medium.Growth medium consisted of 7% normal chicken serum, 8% horse serum, 1076 tryptose phosphate broth and 80% modified Earle's saline plus an additional 0.31% NaHC03. Cells.Chick embryo cells were prepared from decapitated whole chick embryos according to the method of Dulbecco (6) and 25 X lo6 cells in 10 ml of growth medium were placed in petri dishes. After a 48-hour incubation period the cell ,layers were washed with phosphate-buffered saline (PBS) , inoculated with test virus and returned to the incubator for a one-hour virus adsorption period. Unadsorbed virus was then removed and the cell layers covered with 10 ml of maintenance medium and reincubated. All tissue cultures were incubated at 37OC in a humidified atmosphere (RH 9@95%) containing 10% CO,. Other cell layers were treated in a similar manner. Plaque formation was established after an appropriate incubation time, usually 3 to 5 days, by addition of either 0.007% neutral red or 0.1 5 % of 2 (piodophenyl) -3-(p-nitrophenyl) -S-phenyltetrazolium chloride (INT) . Hemadsorption tests were developed by washing the infected monolayers with PBS after removal of the soft agar overlay and adding 3 ml of a 0.5% suspension of chicken red blood cells (RBC) in PBS. Excess RBC were washed off after 20 minutes and the monolayers checked for areas of adsorbed RBC. Tests for total virus were carried out with liquid maintenance medium. At the end of the desired incubation period the infected cells were scraped into the medium and subjected to 3 cycles of freezing at -70°C and thawing at 37°C. The cell debris was removed by centrifuga-

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