Infection of human embryonic cell cultures with the Rauscher murine leukemia virus.

Abstract

The John L. Smith Memorial for Cancer Research, Chas. Pfizer & Company, Inc., Maywood, New Jersey 07607 Although infection of mouse cell lines with murine leukemia and sarcoma viruses can be achieved with relative ease (4, 6, 12), similar infection of human cell cultures has not been reported until very recently. Boiron et al. (2) have described the successful infection of a diploid human embryonic lung culture, WI-38, with the murine sarcoma virus Moloney strain (MSV-M). Infection and continuous replication of animal oncogenic RNA viruses in human cells is of considerable interest as the phenomenon may be a step towards proving the viral etiology of human leukemia. This report describes the infection of human embryonic cell cultures with the Rauscher murine leukemia virus (RLV) and some of the physical and biologic properties of the virus synthesized. Monolayer cultures from the 4th transfer of human embryonic muscle cells, G8-6, growing in RPMI medium #1629 supplemented with 20% fetal calf serum were washed with fresh medium. One ml of mouse plasma virus, HL-67-4 (Rauscher mouse plasma virus supplied by Hazleton Laboratories) diluted 1:4 in growth medium was added to each monolayer in 30-ml plastic tissue culture flasks, and the cultures were incubated at 37~ for 30 minutes. After virus adsorption, the monolayers were washed once and refed with the growth medium. Cytopathic effects were not observed, and after one week the cultures were transferred to 250-rnl Falcon tissue culture flasks. Electron microscopic studies of the infected cells did not reveal the presence of virus at that time; however, after four weeks, during the 17th passage, virus particles were seen budding from the plasma membranes (Fig. 1A) as well as into cytoplasmic vacuoles (Fig. 1B). Virus particles found in the extracellular spaces (Fig. 1C) had the morphology of the C-type particles found in the murine leukemias (1). In subsequent passages, many virus particles were seen by the negative stain technic (3) in concentrates of the infected tissue culture fluids. Extracellular virus in medium from a 5-day-old culture in its 25th transfer since infection was concentrated by 1This study was conducted under contract No. PH 43-66-98 within the Special Virus Leukemia Program of the NIH, USPHS, Bethesda, Maryland 20014. Received May 5, 1969; accepted June 13, 1969. ultracentrifugation in a Sharples continuous flow centrifuge at the rate of one liter per hour (approx. 55,000 X g). The virus was resuspended in either fresh medium or 0.05 M sodium citrate, pH 6.8, to give a 100-fold concentrate. These preparations, which were estimated by electron microscopic studies to have approximately 5 X 109 virus particles per ml, were used for mouse inoculation, serologic tests, and infection of other cell cultures in an effort to characterize the virus. Sixty newborn and ten weanling BALB/c mice were inoculated intraperitoneally with 0.1 ml of this virus preparation. At the present time, three months postinoculation, none of the mice has shown any of the typical Rauscher virus disease symptoms (8). Previous experience with equivalent doses of Rauscher virus derived from infected mouse cell cultures has shown that leukemia should develop within one to two months (5, 10). After six months these mice will be challenged with a Rauscher mouse plasma virus to determine their immune status. Complement fixation tests were conducted using 100-fold concentrates from control and virus-infected cultures as antigens. The antiserum was prepared in rats against mouse-derived virus according to the procedure described by Hartley et al. (5). A titer of 1:16 was obtained against four units of antibody with the positive control RLV that was produced by infected JLS-V9 cells (11). In contrast, the human cell-derived virus concentrate, which had the same virus count, gave a 1:4 titer. The uninfected human cell culture concentrate was negative in this test. The four-fold difference in titers between the two antigens, which was confirmed by a repeat test, would seem to indicate a modified nature of the virus produced in human cells. A potent rabbit anti-RLV serum prepared against mouse plasma virus was used for the immunodiffusion tests. While ether-treated virus from the infected human cells produced precipitin lines, the intact virus showed a negative reaction. The Rauscher leukemia virus produced in mouse cells (JLS-V9), whether ether-treated or intact, was always positive in these tests. The electron microscopic agglutination test (7) was also negative for the intact human cell-derived virus. Further characterization of this virus was attempted by density gradient centrifugation. Two ml of a 50-fold virus concentrate were layered on top of a preformed sucrose gradient. This gradient, along with a similar one containing the 1886 CANCER RESEARCH VOL. 29