A Sedimentable Component of Allantoic Fluid and Its Relationship to Influenza Viruses

Abstract

Macromolecular material was isolated from normal allantoic fluid by a centrifugation procedure comparable to that currently employed for the concentration and purification of influenza viruses. The yield of material was found to vary with the age of the embryo, reaching a maximum average value after 14 days of incubation at 39°C. of about 0.02 mg. per ml. of allantoic fluid. The purified material was found to contain protein, carbohydrate, and lipid and to have a general composition similar to purified preparations of PR8 influenza virus. A typical preparation of normal material had an isoelectric point at pH 2.3. Sedimentation studies indicated that the normal material can give a variety of sedimentation constants depending upon the concentration and viscosity of the preparations. The sedimentation constant, corrected for viscosity, of the major component of a fresh preparation was 170 S. The diameters of the predominant particles shown in electron micrographs of the normal material and of preparations of PR8 influenza virus were about 40 and 100 mµ, respectively. Serological tests indicated that the normal material is a good antigen and that preparations of both A and B types of influenza virus obtained from allantoic fluids by centrifugation show a strong serological relationship to the normal material. Freezing and thawing of allantoic fluid, and repeated adsorption of virus on red cells, failed to provide a practical basis for the separation of normal protein from the virus entity in the case of PR8 virus. In the cases of similar preparations of F12 and of Lee viruses, a partial separation of a small component was accomplished by fractional centrifugation and this component and the normal protein were shown to be identical or very closely related. Antiserum to the purified normal material inhibited red cell agglutination by A and B types of influenza virus at serum dilutions of 600 to 700, but failed to show significant neutralizing capacity in chick embryo and in mouse tests at a serum dilution of 100. Rabbit antiserum to purified preparations of PR8 virus gave a 50 per cent red cell agglutination inhibition endpoint at a serum dilution of 112,000. Some of the implications of the findings are discussed.

Biophysical Properties of Preparations of Pr8 Influenza Virus

“…The results just described as well as the results obtained with preparation 8a in buffer solution provide ample reason for regarding the value of 0.79 as the most probable value for the true specific volume of PR8 influenza virus preparations obtained by differential centrifugation. It can be noted that the composition of this material, as described above and by Knight (25), is not inconsistent with this value for the specific volume.…”

“…The Viscosity of Preparations of PR8 Virus.--Krfight (25) has shown that influenza virus preparations of the type produced in this laboratory contain, in addition to the units presumed to be the virus, considerable amounts of a component indistinguishable by serological means from material isolated from the extra-embryonic fluid of normal chick embryos. The slower component found in preparations 1, la, and 5b at the highest concentrations studied is presumably such a normal constituent.…”

The Nucleic Acid and Carbohydrate of Influenza Virus

“…Preparation 1, but not preparation 2, contained carbohydrate manifestly in excess of that represented by ribose and desoxyribose. This is unusual, for the ordinary contaminant of nucleic acid The results of exploratory color reactions made in this laboratory were interpreted as suggesting the presence in highly purified preparations of influenza virus of both ribonucleic and desoxyribonucleic acids (3,4). Elsewhere, however, the results of similar tests were taken as evidence for the presence of desoxyribonucleic acid and the absence of ribonucleic acid (1,6,21).…”

“…The preliminary treatment of the material to b¢ tested with 10 per cent sulfuric acid constitutes an unusual and probably unwarranted modification of the Bial test (22). Positive Bial reactions were obtained with the unaltered test in both laboratories (3,4,6) and it is now agreed by both groups that this can be taken as an indication that ribouucleic acid is possibly present (23).…”

“…In part the present report describes the isolation of, and the results of tests made on, the nucleic acid of PR8 influenza virus. 1 Investigations on highly purified preparations of influenza virus have revealed the presence of an amount of carbohydrate apparently greater than that accountable for in the nucleic acids (4,6). The nature of this carbohydrate had not been determined, although preliminary tests made byTaylor on the Lee strain led him to suggest the possible presence of mannose or glucose-galactose in complex form, or both (6).…”