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(9) MCBAIN, J. W.,DAWSON, C. R., AND BARKER, H. A,: J. Am. Chem. SOC.66.1021 (1934). (10) MCBAIN,J. W., AND LIU,T. H . : J. Am. Chem. SOC.bS, 59 (1931). R . L., AND RITTER, G. J . : J. Am. Chem. SOC.62, 19.58 (1940). (11) MITCHELL, H., AND CATHCART, W. H.: J. Am. Chem. Soc. 67,1791 (1935). (12) MOUOUIN, (13) NEURATH, H.:J. Am. Chem. SOC.61, 1842 (1939). J. H., AND ANSON, M.L . : J. Gen. Physiol. 12.543 (1929). (14) NORTHRUP, (15) ONSAGER, L,: Phys. Rev. 40, 1029 (1932). J . : J. phys. radium 7, 1 (1936). (16) PERRIN, (17) POLSON, A,: Kolloid-2. 88, 51 (1939). (18) POLSON, A,: Nature 157, 740 (1936). (19) RITTER,G.J . : Ind. Eng. Chem., Anal. Ed. 1, 52 (1929). G. J., AND KURTH, E. F.: Ind. Eng. Chem. 26,1250 (1933). (20) RITTER, (21) STAUDINGER, H.: Trans. Faraday SOC.29, 26 (1933). H.:Papier-fabr. 36, 381 (1939). (22) STAUDINGER, (23)SVEDBERG, T . : Proc. Roy. SOC.(London) Bl27, 1 (1939). (24) V ~ BECKUM, N W.G., AND RITTER, G. J.: Paper Trade J. 106,127 (1937). (25) WHITE, E. V.:J. Am. Chem. SOC.64, 302, 2836 (1912).
SEDIMENTATION CONSTANTS OF PURIFIED PREPARATIONS OF STRAINS OF INFLUENZA VIRUS1$* W. M. STANLEY
AND
M. A. LAUFFERJ
Departmenl of Anamal and Plant Pathology, The Rockefeller Institute f o r Medical Research, Princeton, New Jersey Receaved August 8 , 1946 I~TRODUCTIO~
During the course of a comprehensive investigation of influenza virus, carried out xith a view to the development of useful vaccines (7), a study was made of the biophysical properties of purified influenza virus preparations obtained by means of differential centrifugation (3). Electron micrographs of preparations of the PR8 strain showed slightly irregular, spherical particles with an average diameter of 115 mp. The sedimentation rate was found to vary inversely with the concentration of virus, and this mas found to be due to the variation of solution viscosity with concentration. When purified preparations were examined at high concentrations in the ultracentrifuge there \vas observed, in addition to the virus, a more slody sedimenting component. The amount of this component was found to vary from preparation to preparation and from strain to strain of virus. I t was separated from the virus and was found to possess no virus ac1 Presented a t the Twentieth Kational Colloid Symposium, which was held at Madison, Wisconsin, May 28-29, 1946. * The work described in this paper was done under a contract, recommended by the Committee on Medical Research, between the Office of Scientific Research and Development and The Rockefeller Institute for Medical Research. 3 Present address, University of Pittsburgh, Pittsburgh, Pennsylvania.
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tivity (6). However, this material was found to possess a very high viscosity; hence the viscosity of the usual preparations of influenza virus can be explained as being due in large part to the presence of the more slody sedimenting component. Sedimentation studies on influenza virus in sucrose solutions of varying densities showed a non-linear dependence of sedimentation rate upon solvent density, indicating that the density in solution increases with increasing sucrose concentration (3). However, similar studies carried out in bovine albumin solutions showed a linear relationship between rate of sedimentation and solvent density (4). The results indicated that the density of the PR8 strain of influenza virus is about 1.1, and that the virus particles contain about 60 per cent by weight of water (3, 4). Several strains of influenza virus are known (1). Most of these fall into two groups, Type A strains, of which PR8 is a representative, and Type B strains, of which Lee is a representative. Under comparable conditions, the sedimentation constants of the PR8 and Lee strains are about 700 and 800 S, respectively ( 3 , 5 , 7 ) . Since the Type A strains and the Type B strains do not cross-immunize (I),it is necessary to have representatives of both present in a vaccine in order to secure protection against influenza virus strains of these two common types. During studies on the preparation of such vaccines it was noted that the completed vaccine showed only a single sedimenting boundary in the ultracentrifuge, despite the fact that it contained equal parts of Type A and Type B strains with sedimentation constants of about 700 and 800 S, respectively (6). The sedimentation constant of the material in the vaccine had a value intermediate between those of the separate components of the vaccine. Because it seemed unusual for components having sedimentation constants of about 700 and 800 S to yield a mixture showing only a single sedimenting boundary with an intermediate sedimentation constant, further studies of the sedimentation behavior of strains and of mixtures of strains of influenza vinis were carried out. The results of this study are reported in the present paper. EXPERIMESTAL
In order to establish the reproducibility of the sedimentation behavior of different strains of influenza virus, samples of Lee virus, the only Type B strain studied, were obtained from three different lahoratories and used as inocula. Preparations made in this laboratory were found, under comparable conditions, to yield sedimentation constants essentially identical with that of the Lee virus customarily used in this laboratory. In addition, samples of PR8 virus from three different laboratories were found to yield preparations Trhich possessed the same sedimentation constant, under comparable conditions, as that of the PR8 virus customarily used in this laboratory. Other representatives of Type A xirus, such as F12, ITS, and swine, were found to possess sedimentation constants which varied considerably from preparation to preparation. However, when these xere corrected for solution viscosity (3), values essentially identical with that of the PR8 strain were obtained. It appears that the Lee, as well as
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the Type A strains, tends to contain different but characteristic amounts of the low-molecular-weight impurity possessing a high viscosity. Preparations of the F12 strain tend to contain the largest amount of this impurity. The swine, WS, and Lee preparations tend to contain smaller amounts and the PR8 preparations the smallest amount of this impurity Highly viscous material of low molecular weight has been separated and purified from normal allantoic fluid and from preparations of Lee, PR8, and F12 viruses (2, 3, 6). The properties of the materials from the different sources appear to be quite similar. When such materials from normal allantoic fluid or from Lee virus preparations were added to purified PR8 virus preparations, they were found to decrease the sedimentation constant of the PR8 virus. Because of the large effect on the rate of sedimentation which can accompany an increase in viscosity, and because of the fact that different virus preparations can contain different amounts of this highly viscous impurity, it is obvious that it is necessary to correct the sedimentation constants of influenza virus preparations for the viscosity of the solution if results that are directly comparable are to be obtained. The first vaccine used in these studies, which has been described earlier (6), was prepared from Lee, PR8, and Weiss influenza virus materials produced in chick embryos and concentrated and purified by means of two cycles of differential centrifugation. The intrinsic viscosities of the Lee, PR8, and Weiss virus preparations, before incorporation into a vaccine, \?-erefound to be 34.8, 16.1, and 39.9. Since highly purified preparations of PR8 influenza virus have been found to have an intrinsic viscosity as low as 11.3, these results indicate that the three preparations contain impurities possessing a high viscosity. The amount of this impurity appeared to be low in the PR8 virus preparation and somewhat higher in the other two preparations. The sedimentation constants of the Lee, PR8, and Weiss preparations at a concentration of 2.5 mg. per cubic centimeter in 0.05 M phosphate buffer and corrected for solution viscosity (3) wvtre found to be 832, 719, and 700 S, respectively. A formalinized mixture consisting of two parts of Lee virus, one part of PR8 virus, and one part of Weiss virus was found to show a single sedimenting boundary in the ultracentrifuge with a sedimentation constant, corrected for solution viscosity, of 790 S. I n experiments with individual strains the addition of formalin at the concentration usually used in vaccines, 1 part per 2000 or less, was not found to affect the sedimentation constant of the virus. The second vaccine, which was manufactured by a commercial biological company, was prepared in a manner comparable to that of the first vaccine. The Lee, PR8, and Weiss preparations were examined at a concentration of about 4 mg. per cubic centimeter and found to have sedimentation constants, not corrected for solution viscosity, of 755, 620, and 698 S, respectively. The formalinized mixtiire of these components was found to show a single sedimenting boundary with a sedimentation constant, not corrected for solution viscosity, of 699 S. A third vaccine was prepared by another commercial biological company by means of a single cycle of differential centrifugation. The Lee, PR8, and Weiss virus preparations were examined at a concentration of 2 mg. per cubic
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