The concept of an early peak of virus growth, with little or no residual infectivity at the time of tumor development, has important implications for epidemiologic and etiologic studies of tumors in relation to viral infection. If this type host-parasite relation is frequent with oncogenic viruses, studies using tumor cases as the reference point are greatly handicapped.

The extent to which antibody in mouse tissue suspensions appears to interfere with the detection of virus also has important implications for searches for tumor viruses in other species as well as in studies of the virus-tumor relationship in polyoma virus infection. Mouse parotid tumors should provide a useful system for evaluating the efficacy of procedures for dissociating virus from antibody in tissues.

To summarize: The course of development of infectious virus, hemagglutinin, and antibody in various tissues has been studied after inoculation of polyoma virus into newborn mice, weanling mice, and suckling hamsters.

In newborn mice, virus propagated in a wide variety of tissues, reached maximal titers 7 to 14 days after inoculation, and gradually declined thereafter. Highest titers were generally attained in the kidneys. Virus was recovered from the tissues, and particularly from the parotid tumors, for at least 4 to 5 months after inoculation. Antibody developed within 10 days after inoculation, and reached high levels by the 30th day; the high titers of antibody interfered with the detection of virus in parotid tumors by either tissue culture or mouse antibody production tests, giving prozones and erratic endpoints in infectivity titrations.

The infection pattern in weanling mice was characterized by lower titers of virus during the peak phase and prolonged persistence of very low quantity of virus.

In suckling hamsters, virus reached peak titers 4 to 6 days after inoculation and declined rapidly thereafter; tissues were generally noninfectious by the 3d or 4th week after inoculation. Tumors harvested during the 3d week of infection generally contained small amounts of virus, but tumors harvested at 6 to 11 weeks were generally noninfectious. Two passage lines of virus were maintained for 16 to 19 passages in suckling hamsters by transfer of organ suspensions harvested 5 to 7 days after inoculation.

Dr. Stanton (National Institutes of Health): I would like to express admiration for this excellent and extensive biologic and epidemiologic survey reported by Rowe and his associates. We have been making a much more modest epidemiologic survey for polyoma HI antibodies in our own mouse colonies with findings much in agreement with some of those reported by Dr. Rowe.

In a small colony in which polyoma parotid tumors were induced by inoculation of newborn mice with positive tissue-culture supernatant fluids, we found that the untreated control mice in the same room had an incidence of positive HI serums of approximately 60 percent. This is to be compared with an incidence of only 19 percent in untreated mice in the larger colony from which these animals were originally derived. In attempting to assess the sources of dissemination of polyoma virus in this larger colony with a 19 percent background incidence in the untreated animals, we have studied some 24 different transplantable tumors which are carried in this area. In approximately one third of these transplanted tumors, the host mice had a significantly higher incidence than the background incidence of positive serology.

Study of these animals before and after transplantation showed that they were usually converted from negative to positive within about 2 1/2 to 3 weeks after transplantation.

The number of animals tested in some of these tumors was rather small because of the difficulty of keeping alive animals with fast-growing tumors. But the need for allowing the tumor to grow 2 1/2or 3 weeks to assess the possible presence of polyoma infection in transplanted tumors is very important; more significant than the positive serology was the fact that in 7 of these 8 tumors in which we attempted to isolate virus by tissue-culture methods, we have to date obtained polyoma virus from 5 of the growths. In similar studies on 7 of the tumors with lower or no greater than background incidence of polyoma serology, no polyoma virus was isolated by tissue culture.

No attempt has been made to assess the extent of virus excretion by animals carrying these transplanted tumors. We suspect that possibly in the first 2 weeks after this artificial inoculation of young weanlings and before antibodies are formed, they may excrete virus in significant amounts into the environment.

I would like to ask Dr. Rowe if he has yet studied the rate of excretion of polyoma virus by mice inoculated with infected tumor.

Dr. Rowe: I have that written down to ask you. We have made very few tests, and they have been negative but, epidemiologically, it is almost certain that some of these mice must be good excreters.

Dr. Eddy (National Institutes of Health): I was interested in the observation that the 82-day-old animals were negative on tissue-culture isolation. I was wondering how many blind passages you made. I have had much more experience with hamsters than with mice, but it is unusual not to be able to recover the agent from a virus-induced tumor in the hamster and also in mice that I have tested, even if the animals were very old, though the virus is not always found on the first passage. Sometimes two or three blind passages in tissue culture are required, as with influenza or many other viruses. I was wondering about the negative result with the 82-day-old mouse.

Dr. Rowe: We made only one blind passage. We would not dare make more than one because of the great increase in risk of contamination with further passages in a contaminated environment.

Dr. Eddy: A significant matter mentioned in your paper that you did not discuss here is concerned with the importance of controls. Since, from the beginning, we noted that the virus spread over the laboratory quickly, we instituted what we refer to usually as Monday controls. Each lot of new cells is incubated in the usual way for 2 weeks, and on Monday morning before any other virus is handled, the fluid from the cultures that are 2 weeks old is transferred to new lots of mouse embryo cells. These are in addition to all the other normal cultures that we carry with every single virus passage. Thus we are able to be certain of two things: (1) We do not have or have not encountered polyoma virus in the mice that we are using, and (2) we have a check on our technique.

Furthermore, I think that in any work with any mouse colony it is important that the animals are uniformly susceptible, since there is less chance of the presence of a contaminating virus.

Dr. Habel (National Institutes of Health): In view of the ease with which mice carrying other types of tumors pick up polyoma virus, it is interesting that we have a transplantable polyoma virus-free tumor that, as far as we know, was originally induced by polyoma virus by inoculation of the agent into newborn mice. This was done by Dr. Eddy. The fibrosarcoma developing in this mouse at 6 months after inoculation has been carried through ten transplants in our laboratory in which we are working with polyoma virus. It has been consistently negative for the virus, and the host animals have shown no antibody. Thus, here is a virus-free tumor which, presumably, was initially produced by polyoma virus and is being carried in the laboratory in which the polyoma virus is present.