Aggregates Of Virus Particles Research Articles

Size and ultrastructure of the H-viruses as determined with the use of specific antibodies

The specific reaction between the various H-viruses and their homologous antibodies can be recognized in the electron microscope by the formation of aggregates which stick to the grid surface; cellular debris which does not react with antibody can be removed by washing. Under proper conditions, the aggregated virus particles tend to be linked by fibrous strands, interpreted as antibody molecules, and to be arranged in linear chains in a two-dimensional manner on the grid. In such preparations, the fine structure of the individual particles can be studied readily at high magnifications. The data presented suggest that the basic capsid architecture for all H-viruses is an arrangement of 32 capsomeres in icosahedral symmetry. There appears to be some difference in size between the various H-virus types. H-1 and HT which belong to the same serotype have an average diameter of 215 Å when the particle is measured from side to side rather than from vertex to vertex of the polyhedron. H-3 and RV, on the other hand, which are serologically related to one another, and HB, which is serologically distinct from any of the other types, are somewhat smaller and average 190 Å in diameter when measured similarly.

Electron Microscopic Observations on the Nature of Vaccinia Virus Particle Aggregation

Summary Aggregation among the particles of crude (unpurified) preparations of vaccinia virus is common. Observations with the electron microscope show, in all cases so far examined, that the frequency of groups is greatest for singles and progressively less for each larger size. When the logarithms of group frequency are plotted against the logarithms of group size, the result is a straight line whose slope is a measure of the degree of aggregation. This linear log-log (LLL) distribution is apparent in virus released from cells by freeze-thaw procedure or by ultrasonic waves. It holds for dispersed virus that has been reaggregated by sedimentation into a pellet and resuspended as well as for virus that reaggregates spontaneously. Plaque titrations reveal its influence on the infectivity of aggregated preparations. Its application to interpretation of radiation survival of aggregated virus is discussed. Ultrasonic waves of 20 kc frequency are shown to produce no damage to fresh individual vaccinia virus particles, in intensities necessary for particle dispersion. Freezing at -60°C, heating at 56°C or ultraviolet irradiation (2537 Å) do not predispose the virus to destruction by 20 kc waves.

BIOPHYSICAL AND BIOCHEMICAL CHARACTERIZATION OF LYMPHOCYTIC CHORIOMENINGITIS VIRUS. 2. PARTIAL PURIFICATION BY DIFFERENTIAL CENTRIFUGATION AND FLUOROCARBON TECHNIQUES.

The infectivity of LCM virus suspended in solutions buffered with Tris(hydroxymethyl)aminomethane and its hydrochloride has been found to be greatly stabilized. This stabilization (studied at 4° and 37° C), however, was markedly dependent on the protein content of the solution. If the protein content became too low (by dilution and/or partial purification of the virus), the Tris effect would disappear, but could be reversed if bovine serum albumin was added before inactivation studies were begun. The sensitivity of the virus to ultrasonic irradiation also followed the pattern established above (i.e. — diluted or purified virus was inactivated to a much greater extent than virus suspended in Eagle's medium plus serum). The data also indicated that sonication could break up aggregates of virus particles.

Localization of viral antigen in narcissus leaves infected with yellow stripe virus, determined by means of a fluorescein conjugated antiserum

By means of a fluorescein conjugated antiserum, local aggregation of antigenic material in narcissus leaves infected with yellow stripe virus was demonstrated in the cytoplasm of epidermal and parenchyma cells. By comparing results obtained by fluorescent-microscopy with those obtained by light and electron microscopy it is reasonable to assume that the antigenic material consists of aggregates of virus particles.

Cytological evidence for the production of plant virus ribonucleic acid in the nucleus

Living cells in hand sections of tobacco plants, recently infected with tobacco mosaic virus (TMV) and tomato spotted wilt virus (TSW), were examined in the living state or fixed and stained to reveal ribonucleic acid (RNA). Ribonuclease was used in conjunction with staining to characterize the RNA. Two forms of TMV and defective mutants of one, the tobacco form, and mild strains of TSW that caused mainly chlorotic symptoms were used. Cycles of change in the nucleolus of infected cells were followed in phase microscopy by the apparent emission of material optically less dense than the nucleoplasm. Images of stained sections indicated that this material was RNA. Emission varied according to the virus isolate examined. The strain TMV-U5 differed from the common tobacco strain, TMV-U1. In recent U5 infections, RNA-staining material was found only in the nucleus, and aggregated whole virus was found outside in the cytoplasm. It was concluded that U5 virus was finally synthesized in the nucleus or near its perimeter. TMV-U1 RNA seemed to originate in the nucleolus and pass into the cytoplasm, where it was found in material from which the X-bodies originated. As these masses circulated in the cytoplasm they acquired in part the staining reactions of whole virus, and small aggregates of virus particles began to appear. Uncombined RNA-containing material came together in X-bodies. The defective mutants of TMV had two kinds of material in the cell, one protein-like that appeared in linear or gel-like aggregates, and the other staining like RNA. The RNA again appeared to arise from the nucleolus and pass into the cytoplasm, sometimes mixing with the virus protein gel. It could be removed by RNase without affecting the underlying structure. TSW also gave images suggesting nucleolar emission of RNA, but all virus material in the cell was RNase sensitive. If whole virus covered by a protein coat was formed, the protein did not protect it from digestion by the enzyme.

Virus Particle Aggregation and the Plaque-Forming Unit

Summary Dilute suspensions of vaccinia virus, prepared in various ways, have been sedimented upon a smooth surface and examined by electron microscopy. The observed aggregation, probably similar to that which exists in plaque titration experiments, is found to involve usually over 60% and often over 90% of the particles. Sonic waves are found to be more effective than repeated freezing and thawing, enzymes or pipetting for dispersal of aggregation among the virus particles. This comparison is done by counting and expressing as a percentage of the total count, the number of groups or potentially active units seen in the pictures. The rate of reaggregation of dispersed suspensions was similarly measured. Plaque titration shows the effects of dispersive agents which increase the percentage of active units (AU) and of plaques as well, whereas the total virus particle count remains unchanged. The observed increase in plaques (84% in one experiment) is less, however, than the corresponding increase in AU. This is attributed to the fact that only a small fraction of the virus particles are capable of producing plaques.

Virus strains of identical phenotype but different genotype.

Virus strains of identical phenotype but different genotype.

Variations in the Properties of Potato Virus X and their Effects on its Interactions with Ribonuclease and Proteolytic Enzymes

SUMMARY: When concentrated by precipitation with acid and salts, or by highspeed centrifugation, potato virus X tends to become insoluble though still remaining infective and serologically active. This greatly complicates purification and no method was found that could be relied upon to give good yields of virus with constant properties. Insolubility is correlated with the aggregation of virus particles to form long threads that become entangled, but it is probable that combination of the particles with some cell constituents is also concerned. Insoluble preparations dissolve slowly when incubated with pH 7·5 borate buffer, and rapidly in the presence of trypsin or chymotrypsin. Both of these enzymes hydrolyse virus X, chymotrypsin being the more effective, but different strains of the virus vary in their susceptibility. Ribonuclease readily hydrolyses the nucleic acid derived from virus X, but seems to have no enzymic action on the active virus. When mixed with the virus, the enzyme combines with it and reversibly inhibits infectivity. At pH 7 the addition of ribonuclease to soluble virus preparations causes loss of anisotropy of flow, a fall in precipitin titre, and the production of an insoluble complex. Incubation at pH 7·5 with borate buffer slowly dissolves the complex and restores the original properties of the virus; the rate of re-solution is increased by the presence of trypsin. Some preparations of the virus were partially decomposed by incubation with borate buffer, and sometimes the rate of decomposition was increased in the presence of ribonuclease.

INFLUENCE OF NITROGEN SUPPLY ON THE RATE OF MULTIPLICATION OF TOBACCO-MOSAIC VIRUS

In a former paper the writer (10) reported experiments which showed that the virus activity of juice expressed from tobacco plants diseased with tobacco mosaic was correlated with the amount of nitrogen supplied to the plants. No conclusions could be drawn from these experiments as to whether this increased activity was due to an increase in the rate of virus multiplication or to some other factor, such as greater accumulation of virus particles over a longer period of time, less destruction of virus, or greater dispersion of aggregates of virus particles. Further studies have now been completed which suggest that the variation in virus activity resulting from a change in nitrogen supply may be brought about by a change in the rate of virus multiplication. Results obtained from several repetitions of the experiments to be described below agree closely with those reported in this paper. Materials and methods

X-RAY AND CRYSTALLOGRAPHIC STUDIES OF PLANT VIRUS PREPARATIONS. III

These papers give an account of an optical and x-ray examination of preparations of plant virus substances isolated by Bawden and Pirie, in particular of those of tobacco mosaic disease. They open with a historical survey of the work, indicating the order in which new phenomena were discovered. The subsequent treatment is divided into three parts: I. Introduction and preparation of specimens. II. Modes of aggregation of virus particles. III. (1) The structure of the particles. (2) Biological implications. Part I, after an historical introduction, describes the method of preparation, from solutions of the virus, of optically oriented specimens of different concentrations. For their examination special x-ray apparatus was developed, in particular cameras working with very low angles and capable of indicating spacings up to 1000 A. In Part III, Section 1 deals with the x-ray evidence on the internal structure of the particles. Even in solution, they have an inner regularity like that of a crystal. Virus preparations are thus in a sense doubly crystalline. Closer analysis reveals that the x-ray patterns are not directly comparable to those of a crystal as many of the reflections do not obey Bragg's law, but can be understood on the theory of gratings of limited size. The structure seems to consist of sub-units of the dimensions of approximately 11 A cube, fitted together in a hexagonal or pseudohexagonal lattice of dimensions-a = 87 A, c = 68 A. Contrary to what earlier observations seemed to indicate, the particle seems to be virtually unchanged by drying and must therefore contain little water. There are marked resemblances with the structure of both crystalline and fibrous protein, but the virus structure does not belong to any of the classes hitherto studied. There are indications that the inner structure is of a simpler character than that of the molecules of crystalline proteins. Part III, Section 2 contains a comparative study of the optical and x-ray examinations of three strains of tobacco mosaic virus, two of cucumber disease virus, two of potato virus X, and the virus of bushy stunt disease of tomato. In the last case x-ray measurement confirmed the deduction from its cubic crystal habit that it was composed of spherical rather than long particles, and showed that these had a diameter when dry of 276 A and were arranged in a body-centred cubic close packing. This single example is sufficient to show that the elongated particle form which gives rise to all the anomalous physical properties of the other viruses studied is of no essential biological importance. The similarity and differences observed between the physical properties of these preparations run closely parallel to their clinical and serological classification. Finally, the biological implications of these results are discussed together with possible applications of the new methods of examination to the study of colloid and biological problems.

ACTIVE IMMUNIZATION OF GUINEA PIGS WITH THE VIRUS OF EQUINE ENCEPHALOMYELITIS

An analysis of the preceding experiments discloses that antiviral bodies are demonstrable not at all or in small amounts in the sera of guinea pigs injected with a quantity of active virus not sufficient to induce immunity against the described intracerebral test for induced resistance. However, neutralizing bodies are found in immune animals, although in low concentration, and are regularly manifested when serum is added to low multiples of infective doses of virus under optimal conditions of time and temperature. Hyperimmune serum, on the other hand, reveals a distinct increase in the amount of antiviral bodies present. Irrespective of the mode of procedure for revealing neutralizing bodies, there does not appear to be any notable difference in the content of such bodies in the serum of animals immunized with active virus or with formolized vaccine in which active virus could not be demonstrated. In other words, the antigenic complexes in active as well as in inactive virus produce similar degrees of antibody reaction. The formolization of virus tissue suspensions, therefore, can be considered as a process whereby the virus is inactivated but the antigenicity of the suspensions is preserved, as is also shown in the preceding paper of this series in tests on tissue immunity. In that article is described the remarkably high degree of tissue immunity which results from injections of inactive virus; now we demonstrate that this resistance is associated with a minimal degree of serum antibody. Finally, the question may well be asked, if practically no antiviral bodies are demonstrable immediately or soon after mixing immune serum and virus, and are recognizable in a tenfold increase when functions of time and temperature are brought into play, whether the bodies are "neutralizing" or the phenomenon is due merely to aggregation of virus particles by the serum. From the recent work on the same virus and immune serum (9) by Merrill, there appears to be warrant for the belief in aggregation of virus particles which in turn diminishes the virus activity to the indicated degree.