scholarly journals THE PREPARATION OF HIGHLY PURIFIED PR8 INFLUENZA VIRUS FROM INFECTED MOUSE LUNGS

1946 ◽  
Vol 83 (1) ◽  
pp. 11-24 ◽  
Author(s):  
C. A. Knight
Keyword(s):  
Influenza Virus ◽  
Infected Mouse ◽  
Allantoic Fluid ◽  
Red Cells ◽  
Spherical Particles ◽  
Chick Embryos ◽  
Sedimentation Constant ◽  
Mouse Virus ◽  
Combination Of Methods ◽  
Mouse Lungs

Highly purified preparations of PR8 influenza virus were obtained from perfused, infected mouse lungs by a combination of methods involving adsorption of the virus on and elution from chicken red cells and differential centrifugation. Such preparations were found to possess 50 per cent infectivity end-points at 10–11 to 10–11.8, and 10–13 to 10–14.3 gm. of nitrogen in mice and in chick embryos, respectively. A sedimentation constant of 683 S was obtained for the material and electron micrographs revealed essentially spherical particles about 100 mµ in diameter. The material was isoelectric at pH 5.4 and chemical analyses on several of the preparations indicated the presence of 10.1 per cent of nitrogen, 1.06 per cent of phosphorus, 6.2 per cent of carbohydrate and about 33 per cent of lipid. Positive tests were obtained for both ribonucleic and desoxyribonucleic acids. The virus was precipitated strongly by antisenim to purified PR8 virus obtained from the allantoic fluid of infected chick embryos and this serum inhibited the agglutination of red cells by the mouse virus to a dilution of about 40,000. In general, the properties of the virus isolated from infected mouse lungs were found to coincide with those of the virus obtained from the allantoic fluid of infected chick embryos.

scholarly journals THE SIZE OF INFLUENZA VIRUS

1944 ◽  
Vol 79 (3) ◽  
pp. 267-283 ◽  
Author(s):  
W. M. Stanley
Keyword(s):  
Molecular Weight ◽  
Influenza Virus ◽  
High Molecular Weight ◽  
Particle Diameter ◽  
Allantoic Fluid ◽  
Sucrose Density Gradient ◽  
Chick Embryos ◽  
Sedimentation Behavior ◽  
Virus Activity ◽  
Sedimentation Constant

The sedimentation behavior of influenza virus in dilute solutions of electrolyte was found to be quite variable. At times the virus activity appeared to sediment at a rate comparable with that of particles about 80 to 120 mµ in diameter, at other times at a rate comparable with that of particles about 10 mµ in diameter, and at still other times the bulk of the activity appeared to sediment at a rate comparable with that of the larger particles and the residual activity at a rate comparable with that of the smaller particles. However, in the presence of a sucrose density gradient, the virus activity was always found to sediment with a rate comparable to that of particles about 80 to 120 mµ in diameter; hence it appeared that the variable sedimentation behavior in dilute electrolyte solution was due to convection or mechanical disturbances during centrifugation. About 30 per cent of the high molecular weight protein present in the allantoic fluid of chick embryos infected with the F 12 strain of influenza virus was found to consist of a component having a sedimentation constant of about 30 S, and hence a probable particle diameter of about 10 mµ. The residual protein of high molecular weight was present in the form of a component having a sedimentation constant of about 600 S, and hence a probable particle diameter of about 70 mµ. The proportion of the 30 S component in allantoic fluid of chick embryos infected with the PR8 strain of influenza virus was found to be considerably less. The 600 S and 30 S components of F 12 allantoic fluid were purified and separated by differential centrifugation. The purified preparations of the 600 S component were found to possess a specific virus activity from 100 to over 10,000 times that of the purified preparations of the 30 S component, the difference in activity apparently depending only on the degree of fractionation of the two components. The purified 30 S component was found to sediment normally in the presence of 12 per cent sucrose, whereas the small residual virus activity of such preparations was found to sediment in the presence of a sucrose density gradient with a rate comparable to that of much heavier particles. It is concluded that influenza virus activity is not associated with material having a particle diameter of about 10 mµ, but is associated solely with material having a sedimentation constant of about 600 S and hence a probable particle diameter of about 70 mµ.

scholarly journals CENTRIFUGATION AND ULTRAFILTRATION STUDIES ON ALLANTOIC FLUID PREPARATIONS OF INFLUENZA VIRUS

1944 ◽  
Vol 79 (3) ◽  
pp. 301-317 ◽  
Author(s):  
William F. Friedewald ◽  
Edward G. Pickels
Keyword(s):  
Influenza Virus ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Allantoic Fluid ◽  
Spherical Shape ◽  
Soluble Antigen ◽  
Chick Embryos ◽  
Virus Particles ◽  
Sedimentation Constant ◽  
Primary Virus

A synthetic density gradient technique has been applied to the study of the PR8 and Lee strains of influenza virus in the angle centrifuge. The method counteracted convective disturbances and permitted about a fiftyfold improvement in clearing supernatant fluids of virus. Sedimenting boundaries of infective virus particles, hemagglutinin, and complement-fixing antigen were obtained in the angle centrifuge and correlated with boundaries observed optically in the ultracentrifuge. The sedimentation constant of infective Lee virus particles is approximately 800 Svedberg units, while that of PR8 virus is only about 700. On the assumption of spherical shape, these values correspond to approximate diameters of 85 and 80 mµ respectively. These values agree with those obtained by filtration with graded collodion membranes. The concentration of primary virus particles in untreated allantoic fluid preparations of PR8 or Lee virus is of the order of 0.01 per cent. The primary infective particles are identical with the hemagglutinin and the complement-fixing antigen to a large extent. However, allantoic fluid preparations of PR8 virus also show a slightly inhomogeneous group of particles with an average sedimentation constant of 460 S, which are adsorbed by and eluted from red blood cells yet appear to be non-infective. In addition the virus preparations contain a small amount of "soluble antigen" which sediments more slowly than the virus and is not adsorbed by red blood cells. This soluble antigen is probably associated with material which was observed optically in the ultracentrifuge to sediment at rates ranging from very low values up to that characteristic of the primary virus boundary. This distribution of rate makes it seem likely that the material represents disintegrated virus particles. Calculations based on the experimental results obtained indicate that of the order of 10 influenza virus particles are required to produce infection of chick embryos or mice with the PR8 virus. While a comparable number is required with Lee virus for infection of chick embryos, about 10,000 particles are necessary for infection of mice. The ratio of hemagglutinin to red blood cells required to produce 50 per cent agglutination with dilute virus suspensions in the standard test is roughly 1.

scholarly journals AN EVALUATION OF METHODS FOR THE CONCENTRATION AND PURIFICATION OF INFLUENZA VIRUS

1944 ◽  
Vol 79 (3) ◽  
pp. 255-266 ◽  
Author(s):  
W. M. Stanley
Keyword(s):  
Influenza Virus ◽  
Large Scale ◽  
Allantoic Fluid ◽  
Red Cells ◽  
Virus Protein ◽  
Differential Centrifugation ◽  
Freezing And Thawing ◽  
Protein Nitrogen ◽  
Adult Chicken ◽  
Sedimentation Constant

The concentration and purification of influenza virus by means of differential centrifugation in a vacuum type centrifuge, by adsorption on and elution from adult chicken red cells, by elution of the precipitate formed on freezing and thawing of allantoic fluid, by adsorption on and elution from embryonic chick red cells, and by combinations of the first method with each of the three succeeding methods, have been studied. Over-all yields of virus of about 50 to 70 per cent were obtained by these methods and combinations of methods except for somewhat lower yields when adsorption on and elution from adult chicken red cells was employed. However, the purified products obtained by methods involving only the use of red cells or the freezing and thawing technique were found to contain about 80 per cent of non-virus protein. The purified products obtained when differential centrifugation was used either alone or in combination with any one of the other methods were found to be indistinguishable and to consist of a fairly homogeneous component having a sedimentation constant of about 600 S. Such preparations possessed about 22,000 chicken red cell agglutinating units per mg. of protein nitrogen and solutions containing only about 10–14 gm. of the materials gave 50 per cent infectivity end points in chick embryos. The Sharples centrifuge was found to be almost as efficient as the vacuum type centrifuge for the concentration and purification of influenza virus and, because of its larger capacity, is recommended for the preparation of purified virus on a large scale.

scholarly journals Inactivation of Gonadotrophins

1948 ◽  
Vol 1 (2) ◽  
pp. 271 ◽  
Author(s):  
WK Whitten
Keyword(s):  
Influenza Virus ◽  
Vibrio Cholerae ◽  
Allantoic Fluid ◽  
Chick Embryos

Serum gonadotrophin was rapidly inactivated by preparations of receptordestroying enzyme from Vibrio cholerae arid also by allantoic fluid from chick embryos which had been infected with adapted LEE-B strain of influenza virus.

scholarly journals THE QUANTITATIVE DETERMINATION OF INFLUENZA VIRUS AND ANTIBODIES BY MEANS OF RED CELL AGGLUTINATION

1942 ◽  
Vol 75 (1) ◽  
pp. 49-64 ◽  
Author(s):  
George K. Hirst
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Allantoic Fluid ◽  
Red Cells ◽  
Virus Neutralization ◽  
Influenza B ◽  
Logarithmic Scale ◽  
Neutralization Titer ◽  
Cell Agglutination ◽  
Agglutination Titer

1. The agglutination titer for chicken red cells of freshly prepared or carefully stored suspensions of PR8 influenza virus, that is to say virus of maximum pathogenicity, was found to be proportional to the mouse lethal titer of the same preparations. 2. The agglutination titer of infected allantoic fluid procured in a standard way is relatively constant, regardless of the influenza strain used and its pathogenicity for mice. 3. Virus preparations inactivated by heat or storage may retain their agglutinating power. 4. Certain animal sera contain a partially heat-labile factor which, in low dilution, inhibits the agglutination of chicken red cells by influenza A and influenza B viruses. 5. The agglutination inhibition test, using ferret and human sera, gives qualitative data regarding influenza antibodies which are similar to the information obtained on the same sera by means of the virus neutralization test. 6. There is a definite relationship between the agglutination inhibition titer and the virus neutralization titer of a serum. On a logarithmic scale of both variables, this relationship is essentially linear within the range investigated. 7. The agglutination inhibition titer of immune ferret serum is inversely proportional to the amount of virus used in the test.

pH of the Chorio-allantoic Fluid of Chick Embryos Infected with Influenza Virus B.

1945 ◽  
Vol 59 (2) ◽  
pp. 192-195 ◽  
Author(s):  
I. W. McLean ◽  
G. R. Cooper ◽  
A. R. Taylor ◽  
D. Beard ◽  
J. W. Beard
Keyword(s):  
Influenza Virus ◽  
Allantoic Fluid ◽  
Chick Embryos

scholarly journals A STUDY OF CONDITIONS FOR THE OPTIMUM PRODUCTION OF PR8 INFLUENZA VIRUS IN CHICK EMBRYOS

1944 ◽  
Vol 79 (2) ◽  
pp. 173-183 ◽  
Author(s):  
Gail Lorenz Miller
Keyword(s):  
Influenza Virus ◽  
Blood Cell ◽  
Red Blood Cell ◽  
Allantoic Fluid ◽  
Chick Embryos ◽  
Cell Agglutination ◽  
Uncontrolled Factor ◽  
Optimum Production

In order to determine the conditions for the optimum production of PR8 influenza virus in chick embryos, a study has been made of the róles of concentration of virus in the inoculum, temperature of incubation of infected embryos, length of time of incubation of infected embryos, and age of embryos at the time of inoculation. Relative amounts of virus in different preparations were measured indirectly by means of determinations of chicken red blood cell agglutination titers. Frozen infectious allantoic fluid which produced infection in chick embryos at a maximum dilution of 10–7 was employed as a stock inoculum. Best results were obtained with an amount of stock inoculum of 0.1 cc. of a 10–5 dilution, a temperature of incubation of 35°C., a length of time of incubation of 36 to 48 hours, and with embryos brought to 10 or 11 days of age at 37°C. or 9 or 10 days of age at 39°. An uncontrolled factor arising from inherent variations in the properties of different embryos and different batches of embryos was discussed.

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