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 THE PREPARATION AND PROPERTIES OF INFLUENZA VIRUS VACCINES CONCENTRATED AND PURIFIED BY DIFFERENTIAL CENTRIFUGATION

1945 ◽  
Vol 81 (2) ◽  
pp. 193-218 ◽  
Author(s):  
W. M. Stanley
Keyword(s):  
Influenza Virus ◽  
Ultraviolet Light ◽  
Large Scale ◽  
Allantoic Fluid ◽  
Red Cell ◽  
Differential Centrifugation ◽  
Frozen State ◽  
Elution Method ◽  
Better Than

Influenza virus vaccines containing from 1 to 10 mg. of virus materials per cc. concentrated and purified from infectious allantoic fluids by means of one or two cycles of differential centrifugation and inactivated by different treatments have been prepared and subjected to laboratory tests. Suitable inactivation of the virus preparations with retention of full red cell agglutinating activity and immunizing potency in mice was achieved by treatment with minimal amounts of formaldehyde or ultraviolet light. Treatment with phenol or chloroform failed to cause adequate loss of virus activity. Excessive amounts of formaldehyde or of ultraviolet light were found to cause a loss in red cell agglutinating activity and in immunizing potency. Freezing resulted in the immediate loss of red cell agglutinating activity of the formalinized vaccine. Storage of the vaccines in the frozen state was accompanied by a gradual decrease in red cell agglutinating activity. Drying of the vaccines from the frozen state resulted in a loss of red cell agglutinating activity and, in the case of the formalinized vaccine, in a loss in immunizing potency. There appeared to be at least a rough correlation between red cell agglutinating activity and immunizing potency. The immunizing potency and red cell agglutinating activity of a purified formalinized vaccine containing 2 mg. of virus material per cc. were unchanged following 2 months' storage at 4° but were measurably decreased following storage for 2 months at 18 to 25° and at 37°. At equivalent dosages of virus material the immunizing potency of formalinized centrifugally purified virus, of formalinized virus purified by the red cell elution method, and of infectious allantoic fluid was not measurably different. The immunizing potency of a formalinized polyvalent vaccine containing centrifugally purified Lee, PR8, and Weiss influenza virus materials at concentrations of 5, 2.5, and 2.5 mg. per cc., respectively, was found to be essentially the same as that of a similar vaccine prepared commercially. In both cases the protection afforded against the Weiss strain appeared to be better than that against the Lee and PR8 strains. The commercially prepared vaccine is being subjected to clinical tests in man at dosage levels ranging from 0.01 mg. to 10 mg. The latter corresponds to a level approximately 100 times that of infectious allantoic fluid. It was found that the bacterial contamination that frequently accompanies operation on a large scale can be controlled by the addition of one part per 10,000 of formalin plus one part per 100,000 of phenyl mercuric nitrate to the allantoic fluid immediately following harvesting, without affecting the quality of the vaccine. This procedure and the use of virus materials purified and concentrated by a single cycle of differential centrifugation by means of the Sharples centrifuge were found to be suitable for the production of influenza virus vaccines on a large scale. By means of this method influenza vaccines possessing 20 or more times the immunizing potency of infectious allantoic fluid and 10 or more times the immunizing potency of the usual commercial vaccine prepared by the red cell elution method can be manufactured rapidly on a very large scale with considerable ease and efficiency.

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 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.

scholarly journals The release of influenza virus from the infected cell

1954 ◽  
Vol 52 (2) ◽  
pp. 180-188 ◽  
Author(s):  
L. Hoyle
Keyword(s):  
Influenza Virus ◽  
Cell Membrane ◽  
Infected Cell ◽  
Allantoic Fluid ◽  
Dark Field ◽  
Spherical Particles ◽  
Virus Protein ◽  
Cell Cytoplasm ◽  
Cell Protrusion ◽  
Physiological Phenomenon

1. When detached portions of the chorioallantoic membranes of normal fertile eggs are examined in the dark-field microscope spherical and tubular cytoplasmic protrusions are seen to develop from the margins of the cells and to become detached from them. This is probably a normal physiological phenomenon which occurs to some extent in the intact egg.2. Similar cell protrusions occur from chorioallantoic membranes of eggs infected with influenza virus. The phenomenon appears to be more pronounced in the infected egg than in the normal egg and in particular tubular cytoplasmic protrusions are much more frequent in the infected egg than in the normal.3. Tubular cytoplasmic protrusions may fragment into small spherical particles or may contract into filaments. All types of cell protrusion become detached from the cells and undergo contraction in size probably as a result of loss of water. The infective elementary bodies and filaments present in the allantoic fluid of eggs infected with influenza virus are derived from the cells in this way.4. It is concluded that the infective particle of the influenza virus is a fragment of the cell cytoplasm and consists of a closely aggregated mass of virus protein enclosed in cell membrane.The author is indebted to Prof. W. T. Astbury and Dr R. Reed for the electron photomicrographs reproduced in this paper.

EFFECT OF PROPYLTHIOURACIL ON THE IODINATION AND MATURATION OF RAT THYROGLOBULIN

1966 ◽  
Vol 53 (2) ◽  
pp. 271-285 ◽  
Author(s):  
Claude Simon ◽  
Marie Roques ◽  
Janine Torresani ◽  
Serge Lissitzky
Keyword(s):  
Single Injection ◽  
Freezing And Thawing ◽  
Isotopic Equilibrium ◽  
Sedimentation Constant

ABSTRACT The effect of propylthiouracil on the maturation of rat thyroglobulin in vivo has been investigated. Newly iodinated thyroglobulin dimer is labile to freezing and thawing. This observation has been used to interpret the findings in the present experiments. From experiments using rats in isotopic equilibrium with 125I, and treated with propylthiouracil or propylthiouracil and tri-iodothyronine and also given a single injection of 131I, the following conclusions were formulated 1) the appearance of iodinated S12 thyroglobulin monomer is due to the dissociation of labile iodinated thyroglobulin dimer and appears more readily if the dimer is poorly iodinated, 2) uniodinated thyroglobulin dimer is the most probable substrate for iodination in vivo, 3) maturation of thyroglobulin dimer (as shown by increasing sedimentation constant from 16—17 to 19) is accompanied by increasing amounts of iodine in the molecule, 4) it is not possible to say at present if iodination and iodothyronine formation is the cause or the consequence of thyroglobulin dimer maturation, 5) propylthiouracil might inhibit thyroglobulin maturation by decreasing iodine organification.

scholarly journals Development and evaluation of a monoclonal antibody-based competitive ELISA for the detection of antibodies against H7 avian influenza virus

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yuan Li ◽  
Hongliu Ye ◽  
Meng Liu ◽  
Suquan Song ◽  
Jin Chen ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Influenza Virus ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Large Scale ◽  
Operation Time ◽  
Reference Method ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Concentration

Abstract Background H7 subtype avian influenza has caused great concern in the global poultry industry and public health. The conventional serological subtype-specific diagnostics is implemented by hemagglutination inhibition (HI) assay despite lengthy operation time. In this study, an efficient, rapid and high-throughput competitive enzyme-linked immunosorbent assay (cELISA) was developed for detection of antibodies against H7 avian influenza virus (AIV) based on a novel monoclonal antibody specific to the hemagglutinin (HA) protein of H7 AIV. Results The reaction parameters including antigen coating concentration, monoclonal antibody concentration and serum dilution ratio were optimized for H7 antibody detection. The specificity of the cELISA was tested using antisera against H1 ~ H9, H11 ~ H14 AIVs and other avian viruses. The selected cut-off values of inhibition rates for chicken, duck and peacock sera were 30.11, 26.85 and 45.66% by receiver-operating characteristic (ROC) curve analysis, respectively. With HI test as the reference method, the minimum detection limits for chicken, duck and peacock positive serum reached 20, 21 and 2− 1 HI titer, respectively. Compared to HI test, the diagnostic accuracy reached 100, 98.6, and 99.3% for chicken, duck and peacock by testing a total of 400 clinical serum samples, respectively. Conclusions In summary, the cELISA assay developed in this study provided a reliable, specific, sensitive and species-independent serological technique for rapid detection of H7 antibody, which was applicable for large-scale serological surveillance and vaccination efficacy evaluation programs.

scholarly journals REACTIONS BETWEEN INFLUENZA VIRUS AND A COMPONENT OF ALLANTOIC FLUID

1948 ◽  
Vol 88 (4) ◽  
pp. 463-484 ◽  
Author(s):  
Paul H. Hardy ◽  
Frank L. Horsfall
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
Allantoic Fluid ◽  
Partial Dissociation

Evidence is presented which shows that there is a component present in normal allantoic fluid, probably mucoprotein in nature, capable of combining with influenza A virus (PR8), and that following combination between this component and the virus only partial dissociation of the complex occurs. Evidence is also presented which strongly suggests that the component is present in virus-infected allantoic fluid in which it is in part combined with the virus and in part free although altered by viral action. The probability that the component is present as well in highly purified preparations of influenza virus, and its effect upon various reactions obtained with this agent are discussed.

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