322 H. B. ANDERVONT 



were established. The first, designated the "stem line tumor" arose directly 

 from the original tumor. It was carried through 25 serial passages in chickens 

 and at each passage the slowest growing tumor which did not deviate 

 markedly from the other tumors supplied tissue for the next passage. The 

 second line was started from the eighteenth passage generation of the stem 

 line tumor in which a chicken developed an aberrant, slowly growing tumor. 

 This line represented a deliberate effort to establish a virus-free tumor; 

 older chickens were used and the slowest growing tumor of each passage was 

 used to inoculate chickens for the next transfer generation. This line was 

 carried through 13 serial transfers before it was terminated because it failed 

 to grow in the next passage. The third line was started from the eighteenth 

 generation of the stem line in which a chicken developed a relatively fast- 

 growing tumor. Subsequent passages were made from the fastest growing 

 tumor of each transfer generation. 



Each tumor used for passage was assayed for the virus. Extracts of the 

 rapidly growing third line tumors contained relatively large amounts of 

 virus. Extracts of tumors from the first and second lines showed wide varia- 

 tions in virus content but neither line became free of virus. Bryan (1957) has 

 found that all experimental evidence indicates this virus is intimately 

 associated with the biological characteristics of the tumor cells. This, together 

 with the possibility that the Rous virus is a highly infectious and fixed 

 variant of a stem virus, makes improbable its reversion to the masked state. 



c. Multiplication and Assay. Studies of the multiplication or replication of 

 the Rous virus, as with similar studies of other viruses, include not only the 

 increase in virus quantity but also the rate of virus production by infected 

 cells, the quantitative correlation between detectable virus particles and 

 virus activity, and the use of assay procedures to correlate the number of 

 virus particles with increased activity. The chief objective of such investiga- 

 tions is to ascertain the mechanisms of virus carcinogenesis. 



Sanford et al. (1952) used tissue culture techniques to ascertain the cell 

 type capable of supporting multiplication of the Rous virus. The efforts of 

 previous investigators were summarized in detail, beginning with Carrel and 

 Ebeling (1926), who concluded that the monocyte and not the fibroblast was 

 the infected cell, and including the findings of Ludford (1937) and those of 

 Halberstaedter et al. (1941). In both latter studies the conclusions were 

 contrary to those of Carrel in that the fibroblast was the infected cell. 



Sanford and her colleagues conducted a thorough study of the problem by 

 exposing cultures of chicken fibroblasts or monocytes to nitrates containing 

 the Rous virus and, after varying intervals of time, testing the cultures for 

 virus by injecting them into chickens. The virus remained active at 37.5°C. 

 for 4 days in a mixture of horse serum, balanced saline, and dilute chick 

 embryo extract. In cultures of monocytes the virus survived for 11 to 14 days 



