A major development in the recent study of virus tumors has been the demonstration of virus particles in association with tumor cells and the physical identification of ultrastructural cell-virus interrelationships. The findings, obtained primarily by the electron microscopic examination of thin sections, have revealed the most varied aspects of cell-virus interaction concerned with virus synthesis. These are now known (1, 2) to extend through a spectrum of cell involvement from definitive changes associated with the nucleolus (3), with the total nucleus (4), with nucleus and cytoplasm (5), with cytoplasm alone (6-9), and, finally, to the participation of the cell membrane (10, 11) in the formation or shedding of virus from the cell. Such advances have been an achievement not only in the understanding of morphologic relationships but have provided much physical clarification of previously completely obscure biologic phenomena.

Of greater import than the present status of the problem, however, is the promise of fruitful outcome of further investigation of cell-virus relationships at levels increasingly near those of the biochemical reactions concerned with synthesis of virus and influence of the agents on their respective host cells. The problem of attaining these objectives is presently subject to numerous impediments, both technical and biologic. Resolution with the electron microscope, though great, is decidedly limited with the present techniques of fixation and staining. The obstacles are by no means insurmountable, since much effort is being expended with success (12) to extend the range of size and constituents possible for visualization. In addition, as yet but little exploited, is the whole system of cytochemistry, which is certainly applicable at the level of light microscopy (IS, 14) and much of which may be adaptable to electron microscopy (12, 15-17).

The biologic Umitations constitute a problem of a different order. Analysis by electron microscopy is dependent on resolvable ultrastructure

(18) specific to the infected cell. In the same way the outcome of cyto-chemical study is related to the quantitative aspects of distribution and concentration of specific reactant materials in the cell. Some control or alteration of these biologic relationships has been attained, particularly by tissue culture of tumor cells (10) and by special host-virus adaptations

(19) which have greatly enhanced opportunities for the study of such difficult systems as the Rous sarcoma virus (20). The steady progress in this area gives cause for much optimism that, as in the sphere of physical and biochemical techniques, biologic difficulties may be overcome.

A virus-tumor system providing particularly favorable conditions for investigation of virus-host cell relationships is that of the virus-induced avian leukemia, myeloblastosis (21, 22). The myeloblasts of primitive aspects and neoplastic behavior appear in great numbers in the circulating blood of chickens with the disease. Under proper conditions, these myeloblasts from the chicken proliferate at exponential rates for indefinite periods in tissue culture (28). Moreover, treatment of normal bone marrow in vitro results (24) in growth in culture of cells indistinguishable from myeloblasts obtained from the diseased chicken. Concurrent with growth, the cells liberate the etiologic virus into the culture fluid at rates related to cell characteristics and the conditions of culture. Since the cells flourish in fluid suspension, growth can be quantitated by simple cell count. A special advantage for quantitative studies on virus synthesis and liberation of virus is the measurement of absolute numbers of virus particles by estimate of the highly stable adenosinetriphosphatase activity of the agent (25-27).

Of particular importance for the present report has been the observation, by electron microscopy (9), of definitive cell ultrastructures specific and, under some conditions, quantitatively related to infection of the cell and liberation of the virus. It has been observed, also, that these ultra-structures are of a size applicable to study by light microscopy and to well-developed systems of cytochemistry (14). As a result the opportunity has been afforded to apply the techniques of electron and fight microscopy for correlated studies, together with observations with tissue culture, aimed at the clarification of the dynamic aspects of cell response to the agent. Many of the findings encountered in the work have already been described (9, 14, 24). More recent investigations have confirmed and clarified previously somewhat obscure elements in the sequence on relationships in the origin of the specific structures and their bearing on virus formation. These results, obtained by electron and light microscopy, will be discussed in their bearing on the biologic phenomena of tissue culture reported in an associated publication (28).

Materials And Methods

Myeloblasts were obtained for immediate study and for tissue culture from the circulating blood of chickens with myeloblastosis. The virus employed to produce disease was the BAI strain A (28) inoculated intravenously into line 15 White Leghorn chickens (29) at 5 to 10 days of age. The methods used for collecting the myeloblasts from the blood and from tissue culture, as well as the techniques of establishing and maintaining the cells in culture, have been described in another report (23).

Cells for electron microscopy were fixed in 1 percent osmium tetroxide in Veronal acetate buffer, pil 7.4, for 1 hour at 2° C, delrydrated in graded concentrations of ethanol, and embedded in a mixture of methyl and butyl methacrylates in the proportion of 1:4. Sections were cut with a glass knife on a Porter-Blum microtome. Some sections were floated on lead subacetate to enhance the contrast (15, 16). Electron micrographs were taken with RCA microscopes EMU 2D and EMU 3D.

Cytochemical analyses were made on cells from the bone marrow of normal chickens, from the circulating blood of chickens with myeloblastosis, and on cells from tissue culture. This report is concerned with the results of studies on living cells by phase-contrast microscopy and with examination of the same cells after they were stained for adenosinetriphosphatase activity with the Wachstein-Meisel reagent (SO). A small drop containing myeloblasts obtained by centrifugation from blood or directly from tissue-culture fluid was placed on a coverslip, which was dropped onto a slide with a corner projecting over the edge. The size of the drop was such that the spread did not quite cover the entire area of the coverslip, and, consequently, there was enough pressure to flatten the cells. After examination of indexed groups of cells by phase contrast (14), the preparation was plunged into liquid nitrogen for 30 seconds, the slide was removed from the fluid, the coverslip was pried off, and the slide was placed in a tube immersed in liquid nitrogen which was immediately evacuated. Afterward, the tube was transferred to an alcohol-dry-ice mixture, and the preparation was dried at this temperature in vacuo for 1 hour. Evacuation was continued while the tube was taken from the freezing bath and warmed to room temperature. From this stage, the dehydrated cells were fixed at room temperature in formaldehyde vapor for 3 minutes before application of staining procedures, or were stained without other fixation treatment. Staining for adenosinetriphosphatase was carried out essentially as described by Wachstein and Meisel (SO). The substrate was adenosine-5'-triphosphate, and the period of incubation of the preparation was 2 hours.

Results

The work described here was of 3 principal aspects: (1) continued study of myeloblasts of the circulating blood; (2) determination of the progressive ultrastructural changes in virus-associated myeloblasts under different conditions; and (3) refinement of technique and extension of cytochemical studies on myeloblasts and the cells of normal bone marrow. It has been the principal aim to follow the ultrastructural changes in myeloblasts in correlation with the time and varied conditions of culture.