Dorothy Beard

Ultrastructure of viruses of myeloblastosis and erythroblastosis isolated from plasma of leukemic chickens.

Summary Ultrathin sections of the pellets formed by ultracentrifugation of plasmas from birds with myeloblastosis and erythroblastosis have been examined by electron microscopy. In the material from both diseases there were observed structures of similar appearance represented by images of round or oval shape consisting centrally of a dense core of about 40 mμ diameter. These were surrounded by a less dense material and were limited by an outer membrane-like structure with a total particle diameter of about 80 mμ. In many instances the limiting membrane appeared to be double. The ultrastructure of these agents of the avian leukemic diseases was very similar, if not identical, to that of the viruses of other chicken neoplasms. No particles of these characteristics were obtained from the plasmas of apparently healthy chickens.

Study of the Papilloma Virus Protein with the Electron Microscope.

Information relative to the physical character of the papilloma virus protein 1 has been obtained by means of ultracentrifugation, 2 electrophoresis, 3 , 4 diffusion and viscosity. 2 Recently, more direct studies of this animal virus have been undertaken with the electron microscope. In the present paper are described the preliminary observations dealing chiefly with data gathered with this instrument relative to the appearance and size of the virus. Papilloma virus protein isolated in 0.05 M phosphate buffer pH 6.5 by previously described ultracentrifugal procedures was diluted with water to concentrations of the order of 0.05 to 0.5 mg per cc. The salt concentration in the final solutions was approximately 0.005 M. Preparations for study were made by pipetting the solutions onto collodion membranes supported on 200-mesh wire gauze. Free fluid was removed with the pipette, and the resulting thin film allowed to dry in the air. For comparison, examinations have been made also of mixtures of papilloma and tobacco mosaic viruses† the latter in concentration of about 0.4 mg per cc of the mixture. The essential findings are illustrated in Figs. 1 and 2. In Fig. 1 are shown the results obtained with papilloma virus alone. Repeated micrographs of 3 different batches of the purified protein have resulted consistently in observation of the circular images shown in Fig. 1. When the protein concentration was kept in the region of approximately 0.1 mg per cc, the images were for the most part single, though grouping was frequent as seen in Fig. 1. In preparations of higher concentration, large groups of particles occurred and fewer single images were seen. A regular arrangement of images in the groups was not observed.

Morphology of Characteristic Particles Associated with Avian Erythroblastosis.

Summary A particulate component, which may be the etiological virus, was isolated by ultracentrifugation of the blood plasma of chickens with erythroblastosis. The particles were of spheroidal shape and variable size, averaging approximately 102 mμ diameter. Micrographs of unshadowed preparations showed circular images of an appearance suggesting that the particles consisted predominantly of a watery, low-electron contrast, gellike material surrounding a small relatively dense internal structure. The findings were compared with those obtained in the analogous studies of particles representing the virus of myeloblastosis and that of one form of lymphomatosis.

Dephosphorylation of Adenosine Triphosphate by Concentrates of the Virus of Avian Erythromyeloblastic Leucosis.

Summary A highly potent capacity for the dephosphorylation of adenosine triphosphate has been observed with preparations containing the virus of avian erythromyeloblastic leucosis. This enzymatic reaction occurred with the filtered plasma of birds diseased with the virus and with virus concentrates obtained from the plasma by ultracentrifugal procedures. The partition of enzymatic activity in these preparations has closely paralleled the distribution of virus infectivity of analogous materials measured in other work by titration of the virus in susceptible host chicks. No evidence of the reaction was obtained with the plasma from normal chickens. These experiments demonstrate the specific relationship of the dephosphorylation of adenosine triphosphate with avian erythromyeloblastic leucosis and indicate that the activity is a property of the etiological virus.

Particulate component of plasma from fowls with avian erythro-myeloblastic leucosis.

Summary A particulate material has been isolated by ultracentrifugal procedures from the filtered plasma of chickens diseased with avian erythro-myeloblastic leucosis. The particles were either spheroid in shape and of size varying from 60 to 100 mμ diameter or tailed structures with spheroidal heads and tails of 100 to 200 mμ lengths. Preliminary studies on small amounts of material yielded sedimentation diagrams showing definite, though diffuse, boundaries and an approximate sedimentation rate of 630 S. The ratio of nitrogen to desoxyribonucleic acid of the concentrates indicated a low content of the latter. Material of this sort was not found in the plasma of either normal chicks or chicks refractory to infection with the blood or plasma from birds with avian leucosis. The significance of the component in relation to the etiological agent of avian leucosis is discussed.

BAI strain A avian (myeloblastosis) leukosis virus from myeloblast tissue culture.

Summary A combined culture-dialysis chamber was devised for producing BAI strain A virus in vitro in amounts greatly increasing the scope of physical and chemical studies of the agent. The yield of virus was approximately 15 mg dry wt/day/chamber. Infectivity of the culture virus was the same on a particle basis as that of virus from leukemic chicks. Properties of the culture virus were indicated by gradient centrifugation, gel electrophoresis and by the character of RNA extracted from the agent.

Neutralization and precipitation of the virus of avian erythromyeloblastosis with serum of hyperimmunized chickens.

Summary Chickens have been hyper immunized with formolized concentrates of the virus of avian erythromyeloblastic leukosis together with the untreated plasma of diseased birds containing the agent in high concentration. The resulting immune serums strongly neutralized the infectious properties of the virus and precipitated the characteristic virus particles as observed macroscopically and corroborated by electron micrographs of the precipitates. Precipitation of the virus particles was associated with proportional precipitation of the enzyme activity of the virus to dephosphorylate adenosine triphosphate. The findings constitute the critical specific criterion needed to establish the particles as the virus and the enzyme as a component inseparable, by all methods yet tried, from these virus particles.

Screening of Chicks with Erythromyeloblastic Leukosis for Plasma Activity in Dephosphorylation of Adenosine Triphosphate.

Summary A micro procedure has been designed for the rapid testing, in 5 δ volumes, of the plasmas of chickens with erythromyelo-blastic leukosis for their capacity to dephos-phorylate adenosine triphosphate. Since this enzymatic activity is related both to the number of virus particles and to the infectivity of the plasmas, the test provides the means either for the measurement of those qualities or for the selection of chicks possessing the qualities in the desired degree. There is a wide variation in this enzymatic activity among the individuals of the diseased chick population. For practical application to the study of erythromyeloblastic leukosis, the test is comparable with the hemagglutinative reaction with the influenza virus. The test in its present form would not be useful in the diagnosis of erythromyeloblastic leukosis which can be accomplished much more easily by blood smear. No experiments have been made to learn whether the reaction might occur with the plasma of birds with visceral lymphomatosis which is much more difficult to diagnose in the intact bird.

Identification of Avian Erythroblastosis Virus by Precipitation with Chicken Immune Serum.

Summary Spheroidal particles of about 100-120 mμ diameter occur characteristically in the plasma of chickens with erythroblas-tosis. With formalin-inactivated concentrates of these particles, immune serums have been produced in the chicken which effectively neutralize the infectivity of the agent. These antiserums also cause agglomeration of particles in concentrates identifiable by electron micrography as those found characteristically in this disease. The results have been interpreted to indicate that the characteristic particles agglomerated constitute the viral agent of avian erythroblastosis.

Quantitative Aspects of Micro ATPase Measurements on Plasma from Chicks with Erythromyeloblastic Leukosis.

Summary Changes have been made in the conditions of the micro test of plasmas from chicks with erythromyeloblastic leukosis for their capacity to dephosphorylate adenosine triphosphate. The principal modification has involved use of a balanced saline solution in the reaction mixture resulting in a very large increase in the precision, sensitivity and resolution of the procedure. With 3-λ of plasma, instead of the 5-λ volumes employed earlier, the expected error of a single determination in 2 of 3 cases is ±4 gamma phosphorus on the basis of measurements by electrometric titration. The greater sensitivity has extended the range of plasma activity possible for accurate measurement and has shortened the time necessary for the estimation.