To another point which he made, I agree wholeheartedly that the mammary virus is probably more widely spread than we have realized. In Amsterdam in 1956, I brought up a similar point, and my English colleagues still criticize me strongly for expressing the idea of my chief, Dr. W. E. Gye, of a universal virus. It is not a matter of a universal virus, but it is a fact that the milk virus may be spread in all mouse strains. Some of it may not be present in some and only in small quantities in others, as may be exemplified by findings with the well-known C3Hf strain of Heston. On electron microscopic examination of tumor from several of these mice, we failed to find virus particles, while in others, we found them in small numbers. Biologic tests of some of these failed to show tumor-inducing activity, while others showing few particles by electron microscopy, exhibited very low tumor-inducing activity, about 5 percent. Concentrates of extracts from those tumors that have shown virus particles, when injected into suitable susceptible mice, induced up to 40 percent of tumors.

This is a matter which must be considered with tissue culture studies as well as those in animals. This would be of much interest because of the possible influence of culture medium and temperature on the increase of virus particles demonstrable morphologically and biologically.

Dr. Koprowski (Wistar Institute): Will Dr. Lasfargues and Dr. Dan Moore reply to the comments and statements made by the discussants?

Dr. Lasfargues: It is good to know that we are in agreement with Dr. Muhlbock concerning the influence of hormones on mouse mammary carcinogenesis. Dr. Mtihlbock asked what the milk agent is doing while the mammary gland is in the stage of involution. The milk agent, of course, is present all the time. Baby mice of the C57 strain, for example, initially completely agent-free, become infected by the milk from the mother. The virus particles taken up through the digestive tract are distributed to various organs, including the mammary glands, though not necessarily in the epithelium.

At the time of pregnancy there is a release of hormones that modify the stroma and stimulate the activity of enzyme systems, which modify the properties of the cells and permit penetration of the epithelial cells by the virus.

The virus will multiply there during the time of pregnancy when the mammary epithelium is growing rapidly, and, at the time of lactation, will be released into the milk. In the period of involution many of the cells producing the virus will die, and the virus in them will be lost through the milk. The remaining cells, not being in the proper condition to synthesize the virus, will be in a state of latent infection, and it may not be possible to find any viral particles.

Thus it seems clear that the hormones acting on the epithelium and also on the stroma induce the formation of enzyme systems which are necessary for the synthesis of viral proteins. At all times other than during pregnancy, the virus is latent and is practically not to be seen.

Dr. Eddy asks if it would be possible to condition the tissue culture of one type of cell in roller tubes so that it would be able to support virus synthesis. It is always possible to combine the two types of cells, and it is regarded as a necessary combination. More and more workers have found that a combination of tissues is necessary for the maintenance and multiplication of some viruses. Dr. Algard, for example, in a study of the influence of hormones on hamster kidney tumors, found that the cancer could be induced only if he had a combination of several tissues. He could not obtain the expected reaction if he had cultures of pure cells. But perhaps it would be possible to condition cultures of pure cells by employing tissue extracts, a possibility which we will probably investigate.

The suggestions of Dr. Sabin are appreciated and will certainly be considered. We did not try to filter our supernatant fluids. We only took the tissue of the cultures, ground it with sand in a fluid medium, and centrifuged the extract at a relatively low speed, occasionally at 10,000 rpm, but most of the time at about 1,000 to 2,000 rpm. I do not know if cells were present in the supernatant fluid, but since we were using normal cells absolutely free of virus, we thought that, even if cells were inoculated, the virus would be revealed by bioassay.

Virus of Avian Myeloblastosis. XVI. Kinetics of Cell Growth and Liberation of Virus in Cultures of Myeloblasts

There have been reported from time to time (1-6) the results of studies of tissue cultures of myeloblasts from chickens diseased with the avian myeloblastosis virus. It has been observed that the cells possess the capacity to multiply rapidly under suitable conditions (4-6) and to liberate myeloblastosis virus into the culture medium. Such cells also exhibit pronounced ultrastructural characteristics (8-6) related to intracellular virus synthesis. For many reasons, as will be seen, these cultures of myeloblasts constitute a system of unusual value for the study of virus-host cell relationships with respect to the factors governing the synthesis and liberation of virus and those influencing induction of the state of malignancy. In consequence, much attention has been directed toward the problem of developing conditions of tissue culture suitable for supporting multiplication of the myeloblast.

Until recently, the behavior of the cultures was subject to rather wide fluctuations, with respect both to the rate of cell multiplication and to the rate of virus liberation. As the result of continued investigations, however, there has been devised a culture medium (6) containing growth and nutritional elements greatly effective in the stabilization of the behavior of the cultures over indefinite periods of study. In this system, the rates of cell multiplication and of virus liberation are relatively uniform and predictable within the limits of variation of the potentialities of cells from different individual chicken sources.

It is the purpose of this report to describe some of the more prominent features of cell growth and virus liberation under the best conditions thus far observed. The ultrastructural manifestations of the cells developing in the same and related cultures will be described in an accompanying publication (7).

Materials And Methods

The basic procedures for establishing and maintaining cultures of myeloblasts have been essentially the same since the beginning of the work with these cells (1). Myeloblasts were obtained from the circulating blood of chickens diseased with the myeloblastosis virus designated as the BAI strain A (8). In most of the experiments, the donor birds were White Leghorns of line 15 (9) which are highly susceptible (10) to this agent. These birds respond with a high incidence of disease and, in consequence, afford a relatively high percentage of potential donors for a given study. Chickens of this line, inoculated intravenously at 5 to 10 days of age with doses of about 2 X 10^10 virus particles in 0.1 ml. inoculums, respond within 10 to 20 days with the appearance in the circulating blood of myeloblasts in numbers as great as 2 X 10^6 cells per mm.3 Blood was drawn by heart puncture into tubes containing heparin to prevent coagulation, and the myeloblasts were recovered by centrifugation for 10 minutes at 150 X g. The yield of blood was about 5 to 15 ml., of which the volume of primitive cells (myeloblasts) was about 1 to 4 ml. Donors were selected by the results of daily examination of blood smears.