D. G. Sharp

Enumeration of Virus Particles by Electron Micrography.

Summary A method has been devised for counting virus particles in a suspension by means of the electron microscope. Studies on formolized purified swine influenza virus showed that the particles could be sedimented with a uniform distribution on a collodion membrane in the ultracentrifuge as revealed by electron micrographs of the particles on the membrane. The findings showed a close correlation with the dilution factor and with the number of particles calculated from chemical and physical data obtained with the virus.

Counts of Virus Particles by Sedimentation on Agar and Electron Micrography.

Summary A method has been devised for counting virus particles in electron micrographs of the virus sedimented on an agar surface and removed quantitatively in a collodion pseudoreplica. With influenza virus A (PR8 strain) as test virus, the accuracy of the counts, under conditions practical for routine studies, was the same as that observed with the spray technic or by sedimentation of the agent on collodion. In common with the latter method, sedimentation on agar is applicable to materials of relatively low virus content and, since salt diffuses into the agar, to viruses harmed either by drying in the presence of salt or by salt-free water. The usefulness of this procedure is limited to the study of virus particles which can be recognized either because of characteristic morphology or of other properties that can be related quantitatively to virus behavior.

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.

Rabbit papilloma and vaccinia viruses and T2 bacteriophage of E. coli in shadow electron micrographs.

Summary Electron micrographs of the rabbit papilloma and vaccinia viruses and the T2 bacteriophage of E. coli were obtained with the metal-shadowing technic. The papilloma virus dried in vacuo for application of the metal appeared spheroidal in shape, flattened especially at the region of contact with the film. The vaccinia virus was greatly flattened and showed the presence of a denser internal material bulging beneath the surface. The bacteriophage was a tadpole-shaped structure with a headpiece of well-rounded contours and a stubby tailpiece. The findings were discussed with respect to their bearing on the shape of the viruses in the wet state.

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.

Particulate Component of the Plasma of Fowls with Avian Lymphomatosis.

Summary A particulate material has been obtained from the plasma of chickens affected with the RPL 12 lymphoid tumor strain. Electron micrographs of the particles sedi-mented directly from plasma and of those in ultracentrifugal concentrates reveal spherical or spheroidal particles of sizes varying from 70 to 160 mμ in diameter and an average diameter of about 117 mμ. Particle counts on the individual plasmas of birds with advanced disease gave values of 1 × 107 to 160 × 107 particles per ml in contrast with the normal plasmas in which only occasional or no particles of this size range were observed. The findings with this form of the leukosis complex are remarkably similar to the results of analogous studies on erythromyeloblastic leucosis. The particles seen in the present work are interpreted, tentatively, as representing the virus of one form of avian lymphomatiosis.

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.

The ATPase activity of avian erythromyeloblastic leukosis virus. I. Introductory kinetics.

Abstract An introductory kinetic study of the viral ATPase activity has been based on enzyme particle counts obtained from electron micrographic photographs. This approach has circumvented the necessity of determining enzyme nitrogen, purity and molecular weight. The values obtained for Ks and k30 are 7.34 ± 0.84·10−5 and 0.556 ± 0.022·107, respectively. The close agreement among the values of k30 for five different preparations lends further support for the contention that the virus particle is the enzyme. A value of 5 million has been calculated for the number of molecules of ATP turned over by each enzyme particle per minute. This large turnover number has been discussed.