Inhibition of Influenza Virus Multiplication by Alkyl Derivatives of Benzimidazole I I . Measurement of Inhibitory Actmty by Hemagglutination Titrations

In the foregoing study (1), infectivity titrations were employed to measure the concentration of influenza virus in portions of chorioallantoic membrane in culture medium containing 2,5-dimethylbenzimidazole. This highly sensitive procedure made possible an examination of the effects of this inhibitory compound (2) on the initial phases of multiplication of the virus; i.e., the first reproduction cycle. To secure more information on the mechanism of the inhibitory action of alkyl derivatives of benzimidazole, it was necessary to carry out many experiments involving numerous variables. For this purpose, hemagglutination titrations were employed to measure the concentration of the virus. Because of the relative stability of the hemagglutinating capacity of the virus and the simplicity and precision of hemagglutination titrations, this procedure provided marked advantages. To be able to analyze, with as much precision as possible, the results of experiments on successive cycles of multiplication of the virus and those on the extent of inhibitory activity displayed by a number of alkyl derivatives of benzimidazole, it was necessary to devise a precise technique and control systematic variables closely. During this study it became apparent that variation in the host tissue, i.e. the chorioallantoic membrane of the chick embryo , was a factor of considerable importance, one which affected the amount of virus produced in vitro under strictly controlled conditions. In this communication, the extent to which variation of the host tissue and certain variables in the culture procedure affect the hemagglutination titer of the medium of infected membrane cultures is analyzed. Hemagglutination titer-time curves on the culture medium during multiplication of influenza B virus in portions of chorioallantoic membrane in vitro are described. Experimental conditions are defined under which closely reproducible determinations of the inhibitory activity of chemical compounds can be made relative to influenza virus multiplication. The inhibitory effects of certain selected alkyl 229