Dr. Gross (Veterans Administration Hospital, Bronx, New York): One of the main and very important questions is whether we have a single leukemia virus that is being recovered from mice in different laboratories by various means, or whether we are dealing with a number of viruses.

I think there are some differences between the viruses that we isolated and the one Dr. Moloney described, which will induce leukemia rather indiscriminately in different strains of mice, even in rats. Our attempts with Dr. Woolley to pass the leukemic nitrate into G.W. rats failed. This might have been caused by genetic resistance of that particular breed of rats used in our study.

The leukemia virus that we have been working with in our laboratory, which was originally isolated from AK spontaneous leukemia, is quite selective in its infectivity. It produces a high incidence of leukemia after inoculation into newborn mice of either the C3H/Bi strain, or into newborn mice of strain C57BR/cd. The leukemogenic potency of the agent was much less pronounced when mice of several other strains were used for bioassay, such as those of strains A, C57BL, BALB/c, etc. There is therefore among these two leukemogenic viruses a difference in their ability to induce leukemia in mice of different strains, not necessarily due to concentration of the virus. The leukemic nitrates used in our laboratory have a virus concentration resembling those used by Dr. Moloney.

There is apparently no difference in sensitivity to heat or in stability between these two viruses.

The leukemia virus with which we are working is widely spread in different organs of the injected animals. We found a considerable amount of virus in the blood plasma and a wide distribution in the thymus, spleen, kidneys, and leukemic tumors of mice with virus-induced leukemia. There is, however, only a small amount of the leukemia virus in the brains of leukemic donors.

I was under the impression that we were dealing with a pure lymphatic leukemia virus, since until recently we were able to induce consistently only lymphatic leukemia with this agent. More recently, however, we have observed that among newborn mice inoculated with passage A leukemia virus, and later thymectomized, many developed myeloid leukemia; a few developed chloroleukemia, and some erythroblastic leukemia. Filtrates prepared from such leukemias and inoculated into newborn C3H mice induced in most instances typical lymphatic leukemia. The results of these new experiments present a new problem.

Dr. Melnick (Baylor University): As an animal virologist I, as well as other virologists, almost invariably look for antigenic relationships between viruses even though they may have differing biological properties.

For example, among the ECHO viruses, the prototype Type 9 strain does not propagate in mice. However, a number of other strains have been isolated that do propagate in mice. With some isolates mouse pathogenicity is not a stable property. Upon isolation from nature they may not take in mice, but they can be manipulated in laboratories so they will gain this property. Antigenically, however, the mouse virulent and avirulent Type 9 strains are one and the same agent.

How much work relating to antigenic relationships has been done among these various viruses that produce leukemia? This may be a difficult question to answer, but there are means for its study other than to follow the development of the disease in these animals by performing elaborate cross-neutralization tests. For example, the simple complement fixation test, or other in vitro tests, might reveal relationships among these agents.

Dr. Moloney: Our agent is poorly antigenic. We have initiated some immunological studies in collaboration with Dr. Fink of our virus unit. We have been able to produce what we think may be an effective antiserum in rabbits.

Dr. Bryan: Perhaps Dr. Fink would tell us how the immune serums were produced in the experiments now in progress.

Dr. Fink (National Cancer Institute): The serums that are now under test were prepared in both rabbits and in BALB/c mice. The rabbits were immunized with Dr. Moloney's highly concentrated preparation of virus mixed with Freund's adjuvant. The animals received injections subcutaneously in two sites and were later given a booster injection. Similar preparations made from normal mouse spleens were injected into other rabbits.

The adult BALB/c mice were injected with either formalin-treated or heat-treated virus mixed with Freund's adjuvant. These animals received 2 injections at weekly intervals, either intraperitoneally or subcutaneously. They were then boosted with a very dilute viable virus and bled 3 weeks later. Other mice were inoculated with normal BALB/c spleen extract under similar conditions. Also included in the neutralization tests are normal serums from rabbits and BALB/c mice.

Dr. Bryan: Thank you, Dr. Fink. Dr. Gross, would you care to comment on what serological studies have been made of your virus?

Dr. Gross: The serum was inactivated at 56° C. for 1/2 hour because we found that normal, uninactivated serum neutralized the virus promptly. We also observed, as Dr. Moloney did, that the leukemia virus is not very antigenic.

Dr. Bryan: Dr. Friend, would you care to tell us about your studies?

Dr. Friend (Sloan-Kettering): We have tested by neutralization tests, serums from the mice of Dr. Gross and serums from Swiss mice with leukemic mice of Dr. Schwartz. There is no cross-protection with our agent and these two. Antiserum against polyoma is also ineffective against our leukemia agent.

Unfortunately, the neutralization test is the only serological tool at our disposal. Our agent neither fixes complement nor does it hemagglutinate, and this becomes a very acute problem. But in spite of the fact that there does not appear to be any antigenic relationship at the moment between the seven new viruses, I think we might still be able to attempt a classification, probably by the use of physical and chemical properties that we now know.

Dr. Moloney has made a very nice attempt to correlate all the information available on the seven murine leukemias. It is striking that in morphology in the electron microscope most of these leukemia viruses are similar in size and in shape. It might be interesting to find a number of other characteristics that they have in common, for instance, sensitivity to ether-that is just one I might mention at the moment. I do not know if Dr. Moloney's agent is sensitive to ether.

Another possibility is that if the method that Dr. Moloney uses in concentrating the virus were used to treat other tumors, perhaps we might find or isolate other agents which might be related to some of the seven that we already have.

I would like to ask Dr. Moloney a question about the use of hyaluronidase in his technique. I noted that he not only uses it in the preparation of the tumor, but also in the dilutions for titration. I would like to know what the role of the enzyme is and about the titer of the agent-is it a high-titering agent or a relatively low one, as ours unfortunately is. And I would like to ask another question that has been raised many times before, and which has become more significant since the addition of this new leukemia virus: What is the relationship of the virus to the tumor from which it was isolated? In other words, how can we explain what a leukemia virus is doing in a sarcoma?

Dr. Bryan: Thank you, Dr. Friend. We are going to ask Dr. Moloney to answer these last questions after we hear from Dr. Schwartz. You mentioned that you had tested against antiserum with the Schwartz agent.

Dr. Friend: The antiserum against the Gross agent was rabbit antiserum and the antiserum against the Schwartz agent was not antiserum from rabbits but was from mice that had received the Schwartz tumor.

Dr. Bryan: Would you tell us your story, please, Dr. Schwartz?

Dr. Schwartz (Ilektocn Institute): There are a number of points I would like to clarify. Dr. Moloney referred to our agent as being obtainable from neural tissue. This is quite true, and most of our work has been with brain filtrates, but we are able to get the agent from the tumor as well. Recently we reported with Dr. Sinkovics of our group that one of the reasons we are unable to obtain the agent from tumor tissue in the same concentration and with the same facility as from brain is the fact that there is an inhibiting factor present in tumor tissue extracts which can be separated rather easily from the active virus by a number of techniques.