As a preliminary to a study of the fate of mucoprotein substrate in tissues infected with influenza virus, some characteristics of soluble hemagglutination inhibitors (HI) extracted from chorioallantoic membranes (CAM) have been investigated. The inhibitory material was found to be heat-stable, precipitable with cold ethanol, and subject to progressive inactivation by active viruses or by receptor-destroying enzyme (RDE).

Certain changes in the slope of titration curves obtained with precipitated and non-precipitated fractions upon alcohol fractionation suggested that the HI in heated CAM extracts was heterogeneous. Alcohol in low concentration precipitated preferentially the more efficient (longer?) HI leaving the "weak" component in the non-precipitated fraction. With higher concentrations of alcobol, "strong" HI were converted to "weak" ones, either by denaturation or as result of reduced solubility. These changes in slope of titration curves were reflected in significant discrepancies when densitometric titers (HI50) of fractions were compared with their titers in pattern tests.

The action of active viruses on HI, on the other hand, did not induce qualitative changes in the composition of the inhibitory principle: Titration curves retained parallel slopes even when their position was markedly displaced from that of control curves. In its essential properties, the HI from the CAM appeared to be similar to inhibitors isolated from various other biological sources which have been identified as mucoprotein in nature.

Standards for reproducibility of inhibitory titers obtained by the densitometric method of Hirst and Pickels have been presented.

The relation of HI to the allantoic epithelium has been analyzed. It has been concluded that the HI is a normal constituent and secretion product of these cells. Under physiological conditions, i.e. in the intact egg, the HI contained in the mucoid outer layer of allantoic cells appears to be protected from enzymatic action from without, although adsorption of viral particles may be temporarily impeded. In deembryonated eggs, or in excised membranes, a reduction in total inhibitory substrate as a result of the action of RDE has been observed. It has been shown in experiments on adsorption of active or heat-inactivated virus on the allantoic membrane that prevention of adsorption by RDE may require the synergistic action of the active viral enzyme itself.

It has been concluded that the maintenance or restoration of a normal supply of mucoprotein substrate is a function of the ability of allantoic cells to maintain homeostatic conditions under stress.

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