Precipitation of Active Influenza A Virus from Extra-Embryonic Fluids by Protamine

The allantoic and amniotic fluids of infected chick embryos have been found to contain large amounts of both active influenza virus and the complement fixation antigen 1 , 2 in spite of their low total protein content. These extra-embryonic fluids, either fresh or lyophilized, constitute a very adequate antigen for the diagnosis of influenza by complement fixation test, and the suggestion has been made by us that they should be superior to the usual tissue emulsions as vaccines because of the relatively small amount of cellular debris.

We have carried out a series of experiments to determine the degree of protection afforded to mice by intraperitoneal vaccination with infected extra-embryonic fluids. Five-week-old Swiss mice were inoculated once with 0.05 cc of pooled dialyzed fluids. The fluids used were infectious for mice in dilutions of 10^-8 to 10^-10 and contained about 0.20 mg of N per ml, dialysis having removed the large amount of uric acid nitrogen originally present.

Approximately 3 weeks subsequent to vaccination the mice were inoculated intranasally with 0.05 cc amounts of serial decimal dilutions of egg fluid infected with the homologous strain. In a series of experiments the vaccination resulted in protection of the mice against as much as 10⁸ lethal doses of virus when test inocula of adequate strength were used.

While dialysis eliminates the uric acid, non-dialyzable substances other than virus remain in the fluids in relatively large amounts. The presence of egg proteins can be shown by precipitation with rabbit sera against ovalbumin or whole egg white.

We have attempted to separate the virus from these adventitious substances by various adsorptive and precipitative procedures which will be discussed elsewhere. One practicable procedure of immediate interest consists in precipitation of the active substance by the addition of protamine.