PKOBLEMS CONCERNING THE TUMOR VIRUSES 323 



but did not remain active beyond this period, whereas in cultures of fibro- 

 blasts virus activity continued, for 6| months, at which time the experiment 

 was terminated. They concluded that the fibroblast is probably the host cell 

 of the virus in the tumor. 



Lo et al. (1955) fomid that, in tissue cultures of normal chicken fibroblasts 

 grown in a clot substrate and infected with the Eous virus, the cells became 

 altered within 8 to 14 days. These cytopathogenic effects consisted of 

 degenerating cells and lysis of the clot. Serial subcultures from these degener- 

 ating cultures grew vigorously and appeared healthy for a time, but then 

 hypertrophied cells were produced, of which most disintegrated but some 

 developed into giant cells. All infected cultures were characterized by a 

 variety of cell shapes and sizes, while cells in the noninfected control cultures 

 remained uniform. They also stated: "The morphological alterations in the 

 infected chicken fibroblast cultures were identical with those seen in cultures 

 of Rous sarcoma tissue." 



In view of the recent interest displayed by virologists in tissue culture, it is 

 strange that more have not used it to culture or assay the Rous virus. 

 Manaker and Groupe (1956) showed the possibilities of this approach when 

 they observed discrete foci of proliferative activity in tissue cultures inoculated 

 with the virus; the number of foci was directly related to the amount of virus 

 in the inoculum. Such findings promise a rapid method for quantitative assay 

 of virus suspensions. 



Considerable work is in progress to procure rapid methods for assaying the 

 Rous virus. One promising procedure is based upon the observation by Rous 

 and Murphy (1913) that the virus is capable of inducing hemorrhagic lesions 

 in chickens. This "hemorrhagic disease" was studied intensively by Duran- 

 Reynals (1940a), who found a higher incidence in very young animals, while 

 Milford and Duran-Reynals (1943) showed that chick embryos, when inocu- 

 lated intravenously, developed this lesion instead of tumors. 



Lo and Bang (1955a,b) made a careful study of the factors involved in the 

 occurrence of the lesions in embryos and found their production was favoured 

 by the intravenous route of inoculation, the age of the embryo, and the 

 temperature of incubation. With this information at hand, they developed a 

 method for titrating the virus which was suitable for the determination of 

 relative potencies of virus preparations and for measuring neutralizing 

 antibody against the virus. Borsos and Bang (1957) reported a quantitative 

 relationship between the number of lesions and the amount of virus used as 

 inoculum. 



Development of another assay procedure had its roots in the discovery by 

 Rous and Murphy (1911) that Rous sarcoma cells, or filtrates, produced 

 tumors on the chorioallantoic membrane of the developing chick embryo. 

 Keogh (1938) designed a technique for procuring discrete ectodermal or mixed 



