(CANCER RESEARCH 39. 4322-4329, November 1979] 0008-5472/79/0039-OOOOS02.00 In Vivo Infectivity of the Fibrotropic C-Type Viral Isolates from C57BL/Ka Mice1 Alain Declève,2 Miriam Lieberman, Jacques Boniver,3 and Henry S. Kaplan Cancer Biology Research Laboratory, Department of Radiology. Stanford University School of Medicine. Stanford. California 94305 ABSTRACT It was recently observed (20) that BL/Ka(B) is the only one of the C57BL/Ka fibrotropic viruses able to infect and replicate Of the three fibrotropic C-type viral isolates from C57BL/Ka in vitro in non-virus-producing lymphoma cell lines derived from mice, only the BL/Ka(B) virus is capable of infecting normal individual X-ray-induced tumors of C57BL/Ka mice. It was also hematopoietic and lymphoid cell populations of C57BL/Ka discovered that T + L+ virions may appear in the supernatant mice in vivo, and none are tumorigenic. Inoculation of this virus fluids of BL/Ka(B)-infected lymphoma cell cultures. The hy alone into neonates resulted in transient replication in the bone pothesis was proposed that the thymotropic-leukemogenic marrow, spleen, and occasionally the thymus. Thymocytes functions, absent in BL/Ka(B), had been rescued by this virus could, however, be permanently infected in such animals if BL/ from the nonproducer lymphoma cells and that T—L- viruses Ka(B) were coinoculated with the xenotropic BL/Ka(X) virus. may play a similar role with respect to virus expression in Neonatal injection of BL/Ka(B) prior to fractionated whole- radiogenic lymphomas in vivo. body irradiation yielded an increase in the percentage of virus- In the present report, we have assessed the capacity of the productive radiogenic lymphomas and a decrease in incidence C57BL/Ka fibrotropic isolates to replicate in cell populations of such tumors. Injection of BL/Ka(B) into normal adult C57BL/ present in the lymphoid and hematopoietic tissues of C57BL/ Ka mice did not yield overt expression of virus replication in Ka mice of various age groups, before or after X-irradiation. any of the tissues tested; latent infection could, however, be We have observed that, under certain conditions, BL/Ka(B) is detected in the marrow and in the reticuloepithelium of the capable of infecting and replicating in such cells in newborn thymus. Whole-body X-irradiation of adults with 400 rads par and irradiated adult C57BL/Ka mice and that BL/Ka(N) and tially restored the neonatal susceptibility of bone marrow cells BL/Ka(X) lack this ability. Of particular importance is that the to infection by this isolate. BL/Ka(B) injection after fractionated inoculation of BL/Ka(B) before or after exposure to leukemo whole-body irradiation of weanling C57BL/Ka mice increased genic doses of fractionated whole-body X-rays results in an the percentage of virus-positive lymphomas and revealed that increase of the percentage of radiogenic lymphomas that pro a bone marrow cell subpopulation permissive for infection by duce virus, and allows its detection, in the bone marrow of the virus increases greatly in abundance soon after irradiation. preleukemic animals, of cells that appear soon after the last irradiation and are susceptible to infection by the BL/Ka(B) INTRODUCTION virus. The implications of these findings are discussed. Two major groups of C-type viruses with distinct biological activities have been isolated from C57BL/Ka (Fv-1bb) mice. MATERIALS AND METHODS The first group includes isolates which, like the RadLV" and its in vitro progeny, RadLV/VL3, are T+L + ; they can infect and Mice and Viruses. C57BL/Ka mice of both sexes were transform thymic lymphocytes after inoculation into young adult obtained from our colony and inoculated at various ages i.t. mice of their strain of origin (1, 2, 8, 19, 23). The second group (0.04 ml inoculum in both lobes), i.v. (0.4 ml/inoculum), or i.p. is composed of 2 ecotropic viruses, BL/Ka(B) and BL/Ka(N), (0.1 ml for neonates to 0.3 ml for weanlings), with the following virus preparations: BL/Ka(B), a homogeneous, cloned B-eco- and a xenotropic virus, BL/Ka(X), which, due to their ability to tropic isolate with a titer of 5 x 106 infectious particles/0.4 ml replicate to high titers in fibroblasts of appropriate genotype, on C57BL/Ka mouse fibroblasts (19); BL/Ka(N), a homoge have been designated F + (1, 19). The fibrotropic isolates, neous, cloned N-ecotropic isolate with a titer of 6 x 106/0.4 which are biochemically and serologically distinct from RadLV and RadLV/VLs (1, 2), are devoid of thymotropic or leukemo- ml on NIH/Swiss mouse embryo fibroblasts (19); BL/Ka(X), a xenotropic isolate which has a titer of 1 x 106 infectious genic activity when injected alone or in various combinations into adult C57BL/Ka mice (3). However, these T-L-F+ vi particles/0.4 ml on mink lung cells (19); and RadLV, a leuke mogenic preparation obtained as a 20% cell-free extract of ruses can penetrate the thymocytes of adult animals and en RadLV-induced C57BL/Ka thymic lymphomas (23), which has hance the replication of other viruses which are thymotropic an in vivo titer of 1 x 107 infectious particles/0.04 ml (4). (3). Cell Lines. The SC-1 and mink lung cell lines were obtained 1These studies were supported by Research Grants CA-03352 and CA- through the courtesy of Dr. Janet Hartley, National Cancer 10372 and by Contract NO1-CP-71052 from the National Cancer Institute. NIH. Institute, Bethesda, Md. The BL/RL,?-NP cell line was estab Department of Health, Education and Welfare. lished in our laboratory from an X-ray-induced lymphoma of a * To whom requests for reprints should be addressed. ' Holder of USPHS International Fellowship 5 FO TWO2546; on leave of C57BL/Ka mouse (22). BL/RL,?-NP cells do not produce virus absence from the Department of Pathology, University of Liège. Belgium. or detectable viral antigens, but were found to be permissive * The abbreviations used are: RadLV. radiation leukemia virus; T + , thymo for infection by both RadLV (21 ) and BL/Ka(B) (20). The BL-5 tropic; L+. leukemogenic; F + , fibrotropic; i.t., intrathymic; IF, indirect immuno- fluorescence; TRE, thymic reticuloepithelial. cell line was established in our laboratory from C57BL/Ka Received April 27. 1979; accepted July 24. 1979. mouse embryo fibroblasts (5). NIH/3T3, an NIH/Swiss mouse 4322 CANCER RESEARCH VOL. 39 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1979 American Association for Cancer Research. In Vivo Infectivity of C57BL/Ka Fibrotropic Viruses embryo fibroblast line established by the 3T3 procedure (28), Hematopoietic Tissues at Intervals after Inoculation of was kindly provided by Dr. Stuart Aaronson, National Cancer C57BL/Ka Fibrotropic Isolates. The failure of the T-L-F+ Institute. isolates to replicate in lymphoid and hematopoietic tissues after IF Assays. IF assays for the detection of cytoplasmic viral inoculation into C57BL/Ka weanlings (3) might have been due antigens in cell culture in vitro, and in cell suspensions or to the absence of permissive cells in those tissues at that age. frozen sections from lymphoid and hematopoietic tissues in To test this hypothesis, BL/Ka(B), BL/Ka(N), and BL/Ka(X) vivo, have been reported in detail elsewhere (4, 7, 9). were injected, alone or in combination, and by various routes, Cocultivation Assay with BL/RL12-NP Cells. The cocultiva- into normal or X-irradiated (400 rads) mice of different age tion of BL/RL,?-NP cells with virus-infected cell lines allows the groups. The thymus, spleen, marrow, and in some cases the detection of very small numbers of either RadLV or BL/Ka(B) lymph nodes, were then analyzed at intervals after virus inoc producer cells. Details of these cocultivation experiments will ulation for the presence of IF-positive cells in these organs. No be reported elsewhere.5 IF positivity could be detected even after more than 6 months X-lrradiation. Animals were irradiated at a dose rate of 80 in either normal or X-ray-treated animals that had been inocu rads/min with a 250-kV Philips RT X-ray machine, and cell lated with BL/Ka(N) or BL/Ka(X) at ages ranging from 1 to 48 cultures were irradiated at a dose rate of 1000 rads/min with days (data not shown). In sharp contrast, viral antigens were a Mark I 137Csirradiator. readily detected after inoculation of the B-tropic BL/Ka(B) TRE Cell Cultures. Thymuses were removed from normal virus into C57BL/Ka mice younger than 1 week of age, and in weanlings (1 week after i.t. injection) or from virus-inoculated 6-week-old mice pretreated with 400 rads (Table 1). After animals. The culture technique used6 was a modification of that inoculation of newborns, transient virus replication was de described by Wekerle ef al. (29). The cells were grown on tected in the bone marrow, spleen, lymph nodes, and occa glass coverslips in 60-mm Petri dishes (Lux Scientific Corp., sionally the thymus. The highest number of mice with high Newbury Park, Calif.). By microscopy, such cultures contained percentages of IF-positive cells in the spleen and bone marrow >95% reticuloepithelial cells, less than 5% fibroblasts, and no were found during the first month of life, decreasing sharply detectable lymphoid cells or macrophages. thereafter. Irradiation of adult mice with 400 rads before BL/ To infect TRE cultures, cells were treated for 30 min with Ka(B) inoculation partially restored the susceptibility of bone DEAE-dextran 1 day after subculture and were infected for 2 marrow and spleen cells to virus infection, although the mean hr with 0.4 ml of a 1:10 virus dilution in Polybrene-containing percentage of IF-positive cells was much lower than that ob medium, as previously described (6).