This section is from the book "Symposium Phenomena Of The Tumor Viruses", by U.S. Dept. of Health. Also available from Amazon: Tumor Suppressing Viruses, Genes, and Drugs: Innovative Cancer Therapy Approaches.
In the foregoing description much emphasis has been given to the lack of evidence of virus or material identifiable with virus-derived or associated structures in the myeloblasts in the blood or in culture under certain conditions. Since such cells are not distinguishable in this respect from myeloblasts in the bone marrow of the normal bird, they may be regarded as providing a "control" basis of ultrastructural characteristics suitable for qualitative and quantitative distinction of progressive or regressive change in the dynamics of cell response to the agent in relation to differences in environment. The capacity of the cells to undergo an enormous development of ultrastructural specificity has already been described (9), but there was noted a large variation in the findings not understood at the time. It is now recognized that the differences from one study to another were associated with variation in tissue-culture medium which has recently become subject to control (23, 36). Under these conditions, the characteristics of ultrastructure are much more uniform and predictable.
Myeloblasts from the blood established in a medium of 50 percent chicken serum in medium 199 supplemented with B vitamins, including a high concentration of folic acid (36), exhibit change clearly evident within 2 hours. This is marked, first, by the increase in number, size, and appearance of the structures designated as gray bodies or viroplasts (9). The initial stages in the progression of change are well illustrated in figure 2, which is a section of a myeloblast of the same stock as that of figure 1 after 2 hours in tissue culture. There are now present 3 large gray bodies or viroplasts (PL) of a sort not seen in cells fresh from the circulating blood. In addition, other smaller bodies of somewhat similar appearance are scattered through the section, particularly at the upper right. Such bodies, both large and small, were numerous at this 2-hour interval of culture, but rare after only 1 hour. Nothing else was outstanding, except evidence that some mitochondria presented the appearance of degenerative change (AM). Other and by far the majority of mitochondria were essentially unchanged except for some possible swelling. The structure of the nucleus, nucleolus, Golgi region, and other parts of the cytoplasm showed no identifiable alteration.
During the initial 24-hour culture period, the progression, figure 3, comprises, in part, an increase in the number of viroplasts in the individual cells, practically all of which can be seen thus to be involved. In addition, many of the viroplasts show evidence of virus particles of typical characteristics, as well as bounded circular bodies of the size of the virus, but without the densely stained central region. It should be emphasized that the viroplast structures may differ greatly in size and appearance, a variation of much importance for later interpretations of cytochemical findings. In figure 3 there is a large viroplast of relatively homogeneous staining in which there is a single peripherally located virus particle. Another structure characteristic of the very early cultures is the "blossom" form (9), evident in great profusion in the cell from a 48-hour culture shown in figure 4. These, as seen frequently, contain both typical virus particles and those without dense centers.
It is notable in figure 3 that in the cell of the 2-hour culture there is evidence of extensive mitochondrial alteration (AM), though some of these organelles contain well-defined cristae and membranes. While this cannot be fully ascribed to specific involvement by the virus, there scarcely seems to be any other precursor for the large number of viroplasts present in early culture cells like the myeloblast of figure 4. In this cell there is a broad spectrum of forms of viroplasts (PL) in which well-defined virus particles (VP) are to be seen. It is remarkable to note the numerous mitochondria of entirely normal appearance despite the profound involvement of the total cell and the occurrence of many altered mitochondria.
As the cells are continued in culture in a medium adequate for growth, there is further progression of structural changes associated, in the rapidly growing cell population, with intensification of cell response to the virus. The process of ultrastructural stabilization extends through approximately the 4 th to the 6 th day, to yield cells with the appearance of the cell seen in figure 5 which is a myeloblast after 8 days in the growth medium, i.e., a cell from a culture established 8 days before. After about the 6th day the densely gray viroplasts seen earlier are only rarely present, but instead, there is a variety of forms seemingly representative of a spectrum of structures that range in organizational complexity from mitochondria (AM), which appear to have lost some of their internal regularity, through bodies designated as viroplasts (PL), on to empty vacuoles. In one structure (AM) near the nucleus at the left, there are seen definite cristae at one end and a viroplast-like appearance at the other. This sequence of structural variation is observed for indefinite periods under the conditions described.
The vacuoles vary considerably in size and in the amount of internal stainable material. Some that contain one or more virus particles may be seen near the edge of the cell and may give the impression of being just about to open or of having just opened to the outside. Figure 6 shows an unusually large structure apparently of this nature which contains many virus particles. The inclusion is surrounded by a membrane that seems distinct from the cell membrane but close to it (100-200 A) over a considerable distance. Such a structure containing so many virus particles is but rarely found. Another large group of virus particles lies in the space between the three myeloblasts shown.
An outstanding feature of myeloblasts in culture is their capacity to multiply for indefinite periods under proper conditions (2S). In a medium of 50 percent chicken serum in medium 199 supplemented with vitamins, containing particularly high concentrations of folic acid, the generation rate varies from about 3 to 8 days. It seems remarkable that these cells obviously extensively involved in response to the etiologic agent in the processes of virus synthesis should display such a capacity to engage, at the same time, in the mechanisms resulting in rapid cell multiplication at exponential growth rates. The magnitude of cell involvement is shown by the presence of relatively large numbers of viroplasts and related bodies and, more evident from the dynamic aspects of the relationship, by the prolonged output of virus particles at rates of about 40 particles per cell per hour. Furthermore, and fully as impressive, is the scant evidence in thin sections of damage to heavily involved cells and by the small number of cells taking trypan blue stain in the routine counting of culture cells. In the latter case the number of such elements was less than 5 percent of the total population.
Mitotic figures of normal features were numerous in fixed and stained preparations of culture cells and were seen occasionally in thin sections, as illustrated in figure 7. Here there are evident the centriole pair (C) from which there extend the spindle fibers (SF) toward the disseminated chromatin material (CH). It is seen that numerous viroplasts (PL) of various characteristics are scattered throughout for distribution to subsequently formed daughter cells. Virus particles are discernible in some of the viroplasts during the process of cell mitosis. The cell components, vesical walls, and cell membrane are sharp, though no intact mitochondrion is to be seen in this section.