Goose Erythrocytes Research Articles

Hemagglutination-Inhibition Method and Immunofluorescence Staining with Venezuelan Equine Encephalomyelitis Virus

Hemagglutination and fluorescent antibody (FA) are compared for the direct detection of virus devoid of host cells. A determination was made of the minimal number of tissue plaque-forming units of Venezuelan equine encephalomyelitis virus that could be detected by the hemagglutination technique. Similar concentrations of the virus in bovine albumin borate saline, Brain Heart Infusion broth (Difco), and demineralized water were tested by the FA technique. Somewhat higher concentrations of the virus in bovine albumin borate saline were used in the hemagglutination-inhibition test. The quantitative hemagglutination procedure employed for these studies was carried out at 37 C for 75 min with variations in concentration of goose red cells. As a result of lowering the red cell concentration, smaller concentrations of virus were detected. The direct FA staining procedure applied to slide preparations containing known numbers of tissue culture plaque-forming units of virus was negative. Adsorbed viral antigen on agglutinated goose erythrocytes was visualized by direct and indirect FA techniques.

Studies on the nature of dengue viruses: I. Correlation of particle density, infectivity, and RNA content of type 2 virus

Type 2 dengue virus was harvested from KB cell cultures. The infected culture medium was extracted with fluorocarbon, and virus particles were concentrated and purified by two cycles of centrifugation on layers of fluorocarbon (density 1.574 g/ml). Equilibrium density centrifugation in CsCl revealed two major hemagglutinating components of average densities 1.19 and 1.24. The 1.24 component contained 99% of the residual plaque-forming activity and occasionally could be further fractionated into two subcomponents. Partially purified, P 32-labeled viral HA fractions of different densities were adsorbed on goose erythrocytes, and the virus-RBC complexes were subjected to chemical fractionation. The amounts of label associated with RNA decreased from the densest to the least dense samples and were directly proportional to the PFU titers. Most of the non-RNA label was accounted for in the phospholipid fraction.

Agglutination by Antibody of Erythrocytes Sensitized by Virus Hemagglutinin

Summary When goose erythrocytes are treated with HA of Japanese encephalitis, dengue or Langat viruses, at the optimum pH for hemagglutination, the washed erythrocytes are agglutinable by antibody at pH 7.6. HA is rapidly adsorbed and the sensitized erythrocytes under optimum conditions of preparation may be stored for many days. Nonspecific inhibitor causes slipping of agglutination of sensitized erythrocytes at low serum dilutions. This can be avoided by treatment with kaolin although it is not essential to do so. The sensitized erythrocyte agglutination test is at least as specific as the CF or HAI tests. Diagnostic tests on human sera suggest that the SEA test may have a higher degree of specificity especially if 1-day-old chick erythrocytes are used for preparing antigens.

Reversible inhibition of type 2 dengue virus by agar polysaccharide

Infectivity and hemagglutinating activity of dengue-2 virus are inhibited by a sulfated polysaccharide from agar gel. Inhibition is due to a reversible reaction between virus particles and the inhibitor. Virus-inhibitor complex formation can be demonstrated by sedimenting the inhibitor with the virus particles. Dilution of virus-inhibitor mixtures results in recovery of infectious particles so that pretreatment of virus with inhibitor does not increase inhibition over that which can be accounted for by the amount of inhibitor in the medium at the time the virus is plated. Under conditions of inhibitor excess, the fraction of virus inhibited by a given concentration of inhibitor is constant, i.e., independent of the total virus present. The ratio of virus to inhibitor in the complex has been determined from hemagglutination inhibition tests to be approximately 2 to 1. Presence of inhibitor in culture medium throughout a virus growth cycle does not alter the actual virus yield, but does bind free virus after its liberation from the cell. No evidence for interaction of inhibitor with cells (either KB cells or goose erythrocytes) has been obtained. Comparative studies using other group B arboviruses indicate that these viruses differ in sensitivity to the inhibitor.

Purification, size, and morphology of a mosquito-borne animal virus, Semliki Forest virus

A plaque assay procedure for Semliki Forest virus (SFV) with an efficiency close to 100% has been described. A concentrated SFV preparation of about 97% purity has been obtained from infected suckling mouse brains by a combination of centrifugations and protamine sulfate treatment. The purification procedure results in an increase of more than 1.5 × 10 5-fold in ability to agglutinate goose erythrocytes, with no significant loss of infectivity. The concentration of plaque-forming units of the preparation is 0.9 times that of the virus-size particles, as determined by a count of the particles in microdrops. Sedimentation velocity and electron microscope studies on the particles of the SFV preparation, combined with measurement of infectivity, lead to the conclusion that the virus particles have a sedimentation constant of 320 S and are nonrigid spheres, with a diameter of 50 mμ, or disks. Some of the morphological aspects of SFV particles have been examined in electron micrographs of the suspension and pellet of the virus preparation.

Hemagglutination with arthropod-borne viruses and its inhibition by certain phospholipids

A fluorocarbon extraction technique for the preparation of hemagglutinins from arthropod-borne viruses is described. A number of purified phospholipid fractions prepared from human blood were examined for their power to inhibit the agglutination of goose erythrocytes to virus antigens. A sharp distinction between the behavior of group A and group B antigens was evident. The activity against the group B antigens appeared to be associated with fractions that contained choline, phosphates, and fatty acids.

The Infection of Duck and Goose Erythrocytes by the Mammalian Malaria Parasite, Plasmodium berghei

The infection of mice and baby rats by both Plasmodium lophurae, an avian parasite, and Plasmodium berghei, a mammalian malaria parasite, prompted investigation of the likelihood of P. berghei infecting avian erythrocytes. Though erythrocytes of chick embryos were not infected, those of the goose and duck embryos were. In both these cells the morphology of the parasite was markedly different from that seen in mammalian erythrocytes. Infections were transitory and it was impossible to find parasites after 4 days. Examination of the hosts of both species of parasites showed a rather wide range and examination of the susceptibility of the duck erythrocyte indicated that this cell was peculiarly receptive to infection by a variety of plasmodia.

Some enzymes of isolated nuclei.

THE COMPOSITION OF ISOLATED NUCLEI AND CELL PREPARATIONS FROM TISSUES OF CALF, BEEF, HORSE, AND FOWL WAS STUDIED WITH RESPECT TO THE FOLLOWING COMPONENTS: 1. Liver and kidney arginase, catalase, and uricase; pancreatic lipase and amylase; cardiac muscle myoglobin; erythrocyte hemoglobin; intestinal alkaline phospharase. These are referred to as "special" components in view of their characteristically restricted distribution reflecting the differentiated nature of the tissues in question. 2. Esterase, beta-glucuronidase, alkaline and nucleotide phosphatases, adenosine deaminase, guanase, and nucleoside phosphorylase. These are enzymes of general distribution. The differences in nuclear composition noted with respect to the "special" components, together with the broad variability in nuclear activity found for enzymes of general distribution, led to the conclusion that nuclei are differentiated structures. The following distribution was observed: 1. "Special" components: Hemoglobin was found to be present in fowl and goose erythrocyte nuclei, but myoglobin was entirely absent from heart muscle nuclei; of the special enzymes listed, only catalase and arginase appeared to be concentrated in some of the nuclei. There was no significant nuclear concentration of lipase, amylase, uricase, or alkaline phosphatase. No simple relationship was found between the concentration of a special enzyme in a tissue and its activity in the corresponding nuclei. For example, arginase activity, which is high in mammalian liver and in fowl kidney, was found in liver, not kidney, nuclei. Similarly, catalase activity was demonstrated only in mammalian liver nuclei, although, in mammals, both liver and kidney are rich sources of this enzyme. 2. Enzymes of general distribution fell into three classes: (a) Those present in low concentrations, if at all, in the nuclei-alkaline phosphatase, the nucleotide phosphatases) and beta-glucuronidase. (b) Those present in nuclei in varying concentrations-esterase. (c) Those present in high proportions in most nuclei-adenosine deaminase, nucleoside phosphorylase, and guanase. The exceptionally low nuclear activity of intestinal mucosa with respect to these enzymes was discussed in relation to physiological considerations. The response of nuclei to changes in physiological state was demonstrated by experiments on starvation. The outstanding aspect of this response was a change in nuclear enzymatic activity opposing that observed in the cytoplasm. A comparison of fetal and adult mucosa cells led to the following tentative interpretation of the observed intracellular enzyme distribution: In cells tending to moribundity, as in those subjected to starvation, relative nuclear enzymatic activity falls. The occurrence of special enzymes in nuclei was considered in terms of differentiation, and the high nuclear concentration of the nucleoside-specific enzymes was interpreted in terms of general nuclear metabolic activity.

STUDIES ON BLOOD CELL METABOLISM

1. The respiratory metabolism of non-nucleated mammalian erythrocytes is enormously accelerated and approaches the magnitude of the metabolism of the nucleated erythrocytes of birds on the addition of methylene blue (and certain other dyes), to a final concentration of 0.005-0.0005 per cent. 2. In the presence of methylene blue the respiration is accelerated even when M/1000 KCN is also present. 3. The accelerated respiration due to methylene blue occurs at room temperature but it is most active at 38 degrees . 4. Methylene blue in the above concentration accelerates the respiration of avian (goose) erythrocytes to a much smaller extent than it does the respiration of the erythrocytes of mammalian blood, while the effect upon anemic goose blood seems to be less than it is upon cells of normal goose blood. 5. Owing to a rather large initial carbon dioxide formation in defibrinated blood on incubation, which may not be related to the immediate respiratory process, proper respiratory quotients cannot be obtained in whole blood. When the cells are separated from the serum and suspended in Locke's solution, respiratory quotients are obtained upon incubation comparable to those of other resting mammalian cells, as well as of the actively respiring erythrocytes of birds. 6. The hypothesis is advanced that methylene blue acts in the rôle of an oxygen carrier, supplying a substance which has disappeared from adult mammalian non-nucleated erythrocytes and restoring their metabolic activity to an extent comparable to that of the young immature forms, or to that of the actively respiring avian (goose) blood.