scholarly journals A SEDIMENTABLE COMPONENT OF ALLANTOIC FLUID AND ITS RELATIONSHIP TO INFLUENZA VIRUSES

1944 ◽  
Vol 80 (2) ◽  
pp. 83-100 ◽  
Author(s):  
C. A. Knight
Keyword(s):  
Influenza Virus ◽  
Rabbit Antiserum ◽  
Allantoic Fluid ◽  
Influenza Viruses ◽  
Red Cell ◽  
Serological Tests ◽  
Cell Agglutination ◽  
Small Component ◽  
Serum Dilution ◽  
Neutralizing Capacity

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.

scholarly journals Development of an enzyme-linked immunosorbent assay (ELISA) for the detection of specific antibodies against an H7N7 and an H3N8 equine influenza virus

1984 ◽  
Vol 93 (3) ◽  
pp. 609-620 ◽  
Author(s):  
M. S. Denyer ◽  
J. R. Crowther ◽  
R. C. Wardley ◽  
R. Burrows
Keyword(s):  
Influenza Virus ◽  
Immunosorbent Assay ◽  
Solid Phase ◽  
Allantoic Fluid ◽  
Influenza Viruses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Equine Influenza Virus ◽  
Serum Samples ◽  
Specific Antibodies ◽  
Equine Influenza

SummaryThis paper describes a solid-phase microtitre plate enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to equine influenza viruses. Using egg-grown influenza viruses as the antigens attached to the solid phase, crossreactions were observed between an H7N7 equine virus (designated A1) and an H3N8 equine influenza virus (designated A2) when untreated antisera were tested. Absorption of antisera with egg-grown A/Porcine/Shope/1/33 influenza virus eliminated cross-reactive antibodies so that specific detection of anti-equine influenza A1 or A2 antibodies was possible.Examination of horse sera following vaccination with A1 and/or A2 isolates showed that antibodies were produced against antigen associated with egg allantoic fluid as well as against virus. Such antibodies were eliminated following the absorption of antisera with porcine influenza virus. Results using sera from horses with known vaccination histories confirmed that the ELISA preferentially detected antibodies homologous to the antigen attached to the solid phase and methods to evaluate the current serological state of individual horses by relating the titres of specific antibodies against equine influenza A1 and A2 isolates are shown. This ELISA provides a simple and rapid method of assessing specific antibodies from horse sera and offers advantages over the ‘routine’ HI and SRH assessments since it gives high precision, is economical of reagents and has the capacity to handle large numbers of serum samples.

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 N2Gas Plasma Inactivates Influenza Virus by Inducing Changes in Viral Surface Morphology, Protein, and Genomic RNA

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Akikazu Sakudo ◽  
Naohiro Shimizu ◽  
Yuichiro Imanishi ◽  
Kazuyoshi Ikuta
Keyword(s):  
Influenza Virus ◽  
Plasma Treatment ◽  
Influenza A ◽  
Allantoic Fluid ◽  
High Voltage Pulse ◽  
Influenza Viruses ◽  
Blue Staining ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gas Plasma ◽  
Electron Microscopy Analysis

We have recently treated with N2gas plasma and achieved inactivation of bacteria. However, the effect of N2gas plasma on viruses remains unclear. With the aim of developing this technique, we analyzed the virucidal effect of N2gas plasma on influenza virus and its influence on the viral components. We treated influenza virus particles with inert N2gas plasma (1.5 kpps; kilo pulses per second) produced by a short high-voltage pulse generated from a static induction thyristor power supply. A bioassay using chicken embryonated eggs demonstrated that N2gas plasma inactivated influenza virus in allantoic fluid within 5 min. Immunochromatography, enzyme-linked immunosorbent assay, and Coomassie brilliant blue staining showed that N2gas plasma treatment of influenza A and B viruses in nasal aspirates and allantoic fluids as well as purified influenza A and B viruses induced degradation of viral proteins including nucleoprotein. Analysis using the polymerase chain reaction suggested that N2gas plasma treatment induced changes in the viral RNA genome. Scanning electron microscopy analysis showed that aggregation and fusion of influenza viruses were induced by N2gas plasma treatment. We believe these biochemical changes may contribute to the inactivation of influenza viruses by N2gas plasma.

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 STUDIES ON HOST-VIRUS INTERACTIONS IN THE CHICK EMBRYO-INFLUENZA VIRUS SYSTEM

1949 ◽  
Vol 90 (1) ◽  
pp. 13-22 ◽  
Author(s):  
Werner Henle
Keyword(s):  
Influenza Virus ◽  
Chick Embryo ◽  
Active Agent ◽  
Allantoic Fluid ◽  
Inhibitory Effect ◽  
Influenza Viruses ◽  
Host Cells ◽  
Active Virus ◽  
Homologous Virus ◽  
Host Virus Interactions

Experiments have been reported on the propagation of influenza viruses in the allantoic membrane of the developing chick embryo during the first infectious cycle. After adsorption of the seed virus onto the host cells, only a small percentage of it remains demonstrable by infectivity titrations. This amount remains constant for 4 hours in the case of infection with PR8 virus, and for 6 hours in that of infection with Lee virus. Thereafter, a sharp rise in infectivity occurs 2 to 3 hours before liberation of the new generations of active virus into the allantoic fluid can be detected. Injection of homologous virus, inactivated by ultraviolet irradiation, following infection prevents or delays the production of virus in the tissues, depending to some extent upon the number of ID50 of active virus used as inoculum. The smaller the dose, the more pronounced the inhibitory effect. With increasing delay in the injection of the inhibitor, progressively more virus is produced and liberated 6 and 9 hours after infection with PR8 and Lee virus, respectively. Thus, production of virus may be interrupted by the homologous inhibitor when given up to 3 hours after infection with PR8, and up to4½ hours after infection with Lee virus. Since no increase in infectivity can bedetected during these 3 and 4½ hour periods in the tissues, it is suggested that influenza virus propagates in at least two major stages: first, non-infectious, immature virus material is produced which, subsequently, is converted into the fully active agent. Presumably the first step can be interrupted by the homologous inhibitor, while the second cannot. Heterologous irradiated virus, injected after infection of the tissue, exerts only a slight inhibitory effect on the production of virus.

scholarly journals QUANTITATIVE ASPECTS OF THE RED BLOOD CELL AGGLUTINATION TEST FOR INFLUENZA VIRUS

1944 ◽  
Vol 79 (2) ◽  
pp. 185-195 ◽  
Author(s):  
Gail Lorenz Miller ◽  
W. M. Stanley
Keyword(s):  
Influenza Virus ◽  
Agglutination Test ◽  
Red Cells ◽  
Unit Weight ◽  
Red Cell ◽  
Cell Agglutination ◽  
Quantitative Measurements ◽  
Chemical Purity ◽  
Method Of Measurement ◽  
The Mean

A detailed study has been made of the nature of the variables inherent in the chicken red cell agglutination test for influenza virus in an effort to obtain a method of measurement of biological activity of sufficient accuracy that it might be employed as a reliable index of chemical purity of preparations of the virus. It was found that the temperature at which the test is conducted has a marked effect on the titer, whereas within the range of pH 6–8 the pH has a negligible effect. It was also found that a variation in results may be encountered due to a variation in the specific behavior of red cells from different chickens and to an instability of the red cells themselves. Preparations of purified influenza virus held at 4°C., on the other hand, were found to be stable with respect to chicken red cell agglutinating activity for several months. This fact, together with the fact that in duplicate measurements upon different samples the accuracy was such that the chances were 19 out of 20 that differences of 8.4 per cent in the mean end points were significant, made it possible to establish a reproducible standard of CCA activity based on a unit weight of purified virus material. As a result, it was possible to devise a standardized procedure for carrying out with high accuracy quantitative measurements of influenza virus.

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.

scholarly journals ANTIBODY RESPONSE OF HUMAN BEINGS FOLLOWING VACCINATION WITH INFLUENZA VIRUSES

1942 ◽  
Vol 75 (5) ◽  
pp. 495-511 ◽  
Author(s):  
G. K. Hirst ◽  
E. R. Rickard ◽  
Loring Whitman ◽  
F. L. Horsfall
Keyword(s):  
Influenza Virus ◽  
Antibody Response ◽  
Influenza A ◽  
Geometric Mean ◽  
Allantoic Fluid ◽  
Influenza Viruses ◽  
Human Beings ◽  
Significant Difference ◽  
Antibody Levels

Eleven different preparations of influenza virus were used to vaccinate large groups of human beings. The antibody response to these vaccines was measured by means of the in vitro agglutination inhibition test, and the geometric mean titers of sera taken 2 weeks after vaccination were compared. From these comparisons the following conclusions were drawn: 1. There was a wide individual variation in the antibody response of human beings to the same preparation of influenza virus administrated subcutaneously. The amount of antibody produced by a group with a low prevaccination antibody level was very nearly the same as the amount produced by groups that had higher initial levels. 2. The use of the X strain of distemper virus in the preparation of an influenza vaccine did not enhance the antigenicity of the influenza virus present. 3. Within certain limits the mean antibody response of human beings increased as the amount of virus injected was increased. When large amounts of influenza A virus were given, the antibody response was of the same order of magnitude as that which occurred following actual infection by this virus. 4. When the vaccine was prepared from allantoic fluid, there was no significant difference in the antibody response of human beings given active virus, formalin-inactivated virus, heat-inactivated virus, or virus inactivated by the drying process. 5. Ground infected chick embryos, when diluted with infected allantoic fluid, gave a greater antibody response than allantoic fluid alone (when the virus remained active). The antigenicity of such a preparation was diminished when the virus was inactivated by formalin. 6. Antibody levels 6 and 9 weeks after vaccination showed a marked drop from the 2-week postvaccination levels. In a small group the antibody levels at 5 months were still further reduced. Those individuals who possessed the higher titers tended to lose their antibodies faster than did those at a lower level.

scholarly journals Comparison of the infectivity of influenza Viruses in two host Systems: the Allantois of infect Eggs and Surviving Allantois-on-Shell

1958 ◽  
Vol 56 (4) ◽  
pp. 535-546 ◽  
Author(s):  
S. Fazekas de St Groth ◽  
D. O. White
Keyword(s):  
Influenza Virus ◽  
Virus Particle ◽  
Host Resistance ◽  
Thermal Inactivation ◽  
Allantoic Fluid ◽  
Relative Sensitivity ◽  
Influenza Viruses ◽  
Critical Step ◽  
Test Systems ◽  
Intracellular Stage

It is shown that the assay for infectivity in bits of allantois-on-shell, which has been standardized on the BEL strain, is optimal also for nine representative strains of influenza virus tested.On the average, the sensitivity of the technique is the same as of orthodox allantoic infectivity tests; its precision is always higher. The relative sensitivity of the two tests varies from strain to strain, the log tray/egg differences being SW (+0.88), MEL (+0.51), CAM (+0.31), PR8 (0.28), WSE (+0.25), HUT (−0.02), BEL (−0.22), FMI (−0.35), LEE (−0.47), BON (−1.02). This gradient is the same for fully infective and incomplete forms of influenza virus.The tray/egg gradient of susceptibility is negatively correlated with the variation in host resistance.The differences in susceptibility are not due to any effect of the plastic trays, to thermal inactivation of the virus, to differences in adsorption or viropexis in the two test systems or to the effect of allantoic fluid on the virus particle. The critical step has been shown to occur during the intracellular stage of multiplication.

scholarly journals NEUTRALIZATION TESTS WITH SERA OF CONVALESCENT OR IMMUNIZED ANIMALS AND THE VIRUSES OF SWINE AND HUMAN INFLUENZA

1936 ◽  
Vol 63 (5) ◽  
pp. 645-653 ◽  
Author(s):  
Thomas Francis ◽  
Richard E. Shope
Keyword(s):  
Influenza Virus ◽  
Swine Influenza Virus ◽  
Swine Influenza ◽  
Influenza Viruses ◽  
Human Influenza ◽  
Human Influenza Virus ◽  
Neutralizing Capacity ◽  
Heterologous Virus

Human and swine influenza viruses were regularly neutralized by their homologous immune sera. However, the sera of animals convalescent from infection with either the swine or human influenza virus possessed little, if any, neutralizing capacity for the heterologous virus. Hyperimmunization of animals against swine influenza virus tended to increase the neutralizing capacity of their sera for human influenza virus, but in an inconstant fashion, whereas repeated inoculations with human influenza virus frequently resulted in sera with strong neutralizing activities against swine influenza virus. These observations serve to emphasize both the immunological distinctiveness and the interrelationships of swine and human influenza viruses.

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