I have the feeling, too, that these gray bodies which we have seen only rarely in Rous sarcoma cells might be increased greatly in number if we could improve our tissue-culture medium. Perhaps, then, more could be seen, and they might be found really to correspond to virus formation centers. Up to now our pictures are not absolutely comparable to \-ours in that respect, but this does not mean, however, that the Rous system might not be similar to myeloblastosis if we could improve our methods.

Dr. Palade (Rockefeller Institute): Dr. Bonar, how good is your evidence that what you call "small gray bodies" are the normal precursors of mitochondria?

Dr. Bonar: Our evidence on this point is negligible. We have simply observed that these structures are rather frequent in these cells. If mitochondria are involved in the viroplast sequence and normal mitochondria must be formed as replacements, it seems possible that these are precursors. We have simply taken this idea from the literature, and it is not an essential point of our argument. We are not certain that they are precursors.

Dr. Palade: According to your diagram of the normal cell, the gray body is the precursor of both mitochondria and granules. What granules do you have in mind?

Dr. Bonar: I was thinking of the granules of myelocytes and of mature granulocytes which are derived normally from myeloblasts. There have been two reports in the literature concerning the origin of these granules. In both, formation from mitochondria was suggested. This was based on electron microscope evidence.

Dr. Palade: Do you have any evidence about the enzymatic content of those granules in the mature white blood cell?

Dr. Bonar: Yes, we have, from the application of the staining technique of Wach-stein and Meisel. It appears that in the mature cells ATPase activity is localized in the granules. It might be mentioned, also, that there is some difference in sensitivity to various fixation procedures between the granules of the myelocytes and those of the mature cells. Also, you may have noticed that only some of the myelocyte granules reacted, while almost all the granules of the mature cell were positive.

In the viroplasts in the infected myeloblasts, preliminary studies have been made to isolate the bodies by centrifugation of cell homogenates. It was found, in the electron microscope, that the mitochondria and the viroplasts appear together in the usual mitochondrial fraction. It has not yet been possible to get this fraction in a satisfactory state of purity, since there is always much apparently microsome material present. However, the ATPase activity of this fraction is much higher than that which has been reported for other mitochondrial material, that is, from liver and so on.

Dr. Palade: Is it not possible that the ATP is split by another phosphatase in the granule?

Dr. Bonar: Other substrates have been tested including ADP, AMP, and glycerophosphate, and a little activity was found with ADP. It is difficult to quantitate the reaction very well with these cytochemical techniques, but it appears that the reaction with ADP was less than that with ATP. With AMP and glycerophosphate, there was essentially no reaction.

However, if the cells are damaged by sufficient pressure under the coverslip to cause bursting or extrusion of processes from the cell, the mitochondria may exhibit ATPase activity.

Dr. Palade: Do you visualize, then, that the ATPase activity originally found in the gray body or in the granule is transferred, finally, to the virus particle?

Dr. Bonar: Yes, it is reasonable to suppose that, as the virus particle forms in this environment containing ATPase, it incorporates the enzyme, perhaps only incidentally. It might be noted that the virus particle also contains normal chicken tissue antigen which is probably picked up in the same way.

Dr. Palade: Finally, would it not be possible to use a tracer material visible in the electron microscope to determine whether the vesicles containing the virus particles are opening and releasing the virus particles to the medium or perhaps taking them up from the outside?

Dr. Bonar: It is possible. The plan at present is to examine the cells with phase contrast over long periods for observation of the movement of the bodies. The cells have not been tested for phagocytic activity, but this is also planned.

Dr. Hose (Columbia University): Apropos of the observation Dr. Bonar has made in connection with the possible release mechanism of viral particles from the myeloblast and also of the discussion which followed his presentation, perhaps it would be helpful to indicate that evidence obtained with another virus suggests the operation of the same sort of mechanism.

Dr. Morgan, Dr. Holden, Dr. Jones, and I reported, in the Journal of Experimental Medicine last fall, some observations with herpes simplex virus in which exactly this kind of mechanism was postulated as the result of the electron microscopic observations. I, for one, am convinced from Dr. Bonar's data that this mechanism is operating in the agent he has under consideration, and I have no doubt that this same mechanism probably operates in other viruses.

Do you view the formation of virus as not a destructive process but as the continuing elaboration of the agent by surviving cells? If that is the case, within any one cell, is the formation of virus a synchronized process? Or do you find within a cell, a single-cell, all stages of virus formation?

Dr. Bonar: After the first period of 2 to 4 days in culture, we often find in one cell section a number of different stages of what we believe to be the process of virus production.

Dr. Dmochowski (M. D. Anderson Hospital): Dr. Bonar, have you found in the cells grown in tissue culture the formation of membranes in the cytoplasm that Dr. Haguenau has described as myelin figures and which we call membranous structures?

Dr. Bonar: No, with the possible exception that we see vacuoles or vesicles which do not contain virus particles. Of course, particles may be there, but outside the plane of sectioning. We have seen nothing resembling myelin figures or these multiple membranous components.

Dr. Dmochowski: A comment which I believe is pertinent concerns the satisfaction one feels when he observes changes in tissue culture similar to those seen in the cells of organs of chickens infected with the same type of disease. We have observed similar structures-we do not describe them as gray bodies but as osmiophilic bodies- and the presence of virus particles in mitochondria. It is pertinent, also, to point out that similar changes have been observed by us in erythroblastosis of two strains, RPL12 and the Engelbreth-Holm strain It and, too, in visceral lymphomatosis, but not in Rous sarcoma.

Therefore, I was interested to hear from Dr. Haguenau that a similar trend of events is taking place in Rous sarcoma cells, if I understood her correctly, when the cells are grown in tissue culture.