UNUSUAL VIRUS IN HUMAN TUMOR CULTURES original nasopharyngeal biopsy sample removed in East Mrica. Certainly, the cultures were at all times maintained in human serum, thus excluding the possibility of the inadvertent introduction of an anima virus in serum of animal origin. Although morphologically the virus is indistinguishable from those reported from a number of bovine lymphosarcomas (7), and a feline fibrosarcoma (2), and further resembles these agents in the induction of syncytia (1, 2), such a virus does not appear to have been describeel before in human material. There have been many reports of “C”-type virus particles in material obtained from human malignancies. but, as pointed out by several authors (9-14), the vast majority cannot be regarded as acceptable. In the few cases where unequivocal, budding, "C"type particles were demonstrated in human tumor cells (15-17), these particles are totally different from the present agent in structure and mode of maturation. As indicated, the virus reported here is morphologically indistinguishable from 2 unusual animal viruses (7. 2). It has been suggested (1) that 1 of these resembles the mouse mammary tumor virus. The present virus, and the 2 morphologically similar animal viruses (1, 2), do indeed in some respects resemble the mouse mammary tumor agent in having as their immature forms cytoplasmic ring-shaped “A” particles and a process of maturation by budding at cellular membranes. On the other hand, there are crucial differences: The mouse mammary tumor virus is longer than the new virus. The de novo accumulation of crescentic dense material which usually enters buds of the mouse mammary tumor virus (18) has never been observed with the present virus which invariably matures by a preformed immature spherical particle entering a bud (figs. 6-8). Next, the dramatic radiating spines on the mature form of the present virus (figs. 6-8) are in marked contrast to the fringe of small projections on the mammary tumor agent. Lastly, once released, the present virus does not undergo further stages of maturation such as occur when the ring-shaped core of the mammary tumor virus is rearranged into an eccentric dense nucleoid (18). It would appear that the new agent described here in human tumor material, and the 2 similar animal agents (1, 2), do not entirely fit into any VOL. 46, NO. 2, FEBRUARY 1971 408-613-71-12 301 currently known morphological family of viruses and may represent members of a new group. Such a group should also include simian founy viruses (19), since these have recently been shown to resemble the 3 agents discussed here, both in morphology and morphogenesis (20). The 2 animal examples of this new morphological group were found in association with malignant tumor cells (1, 2). That the present human example has also been identified in material from a tumor may be no more than a coincidence, The virus was not observed in the original biopsy sample of the tumor, in carly fibroblasts grown as a monolayer from it, or in rounded dying cells released from the monolayer after 1 week in culture. With present techniques, viruses of this type possibly survive best when carried as passengers in malignant ceils, and become evident only when such cells grow in vitra. On the other hand, the source of each of these viruses might prove significant and certainly calls for further investigation. Human nasopharyngeal carcinoma is an unusual tumor with a curiously high incidence rate among Southern Chinese and in a small area of Kenya. It is a tumor that has been linked on epidemiological and preliminary experimental grounds to the EB virus (3-5), and it might ultimately prove of viral etiology. The finding of a highly unusual new viral agent in material from such a tumor makes it important to intensify the search for a possible viral cause. Further studies on the agent newly reported here and its relationship with nasopharyngeal carcinoma in East Africa are being pursued. REFERENCES (1) Malmquist W., Van der Maaten MJ, Bone AD: Isolation, immunodiffusion, immunofluorescence, and electron microscopy of a syncytial virus of lymphosarcomatous and apparently normal cattle. Cancer Res 29:188-200, 1969 (2) McKissick GF, LAMONT PH: Characteristics of a virus isolated from a feline fibrosarcoma. J Virol 5:247-257, 1970 (3) OLD LJ, Boyse EA, OetigeN HF, et al: Precipitating antibody in human serum to an antigen present in cultured Burkitt's lymphoma cells. Proc Nat Acad Sci USA 56:1699-1704, 1966 (4) DE-THE G, AMBROSIONI JC, Ho HC, et al: Lympho blastoid transformation and presence of herpes-type viral particles in a Chinese nasopharyngeal tumour 302 ACHONG, MANSELL, EPSTEIN, AND CLIFFORD cultured an astro. Nature (London) 221:770-771, 1969 (5) HENDE W, HENEE G, Hồ HC, et al: Antibodies to Epstein-Barr virus in nasopharyngeal carcinoma, other head and neck neoplasms, and control groups. J Nat Cancer Inst 14:225–231, 1970 (6) Ersiras MA, Acnone BG: Fine structural organizanon of human lymphoblasts of a tissue culture strain (EBI) from Burkitt's lymphoma. J Nat Cancer Inst 34:241-253, 1965 (7) Chandra S: Undulating tubules associated with endoplasmic reticulum in pathologic tissues. Lab Invest 18:422-428, 1968 (8) Chandra S, Moore GE, Brandt PM: Similarity between leukocyte cultures from cancerous and noncancerous human subjects: an electron microscopic study. Cancer Res 28:1982-1989, 1968 (9) Auxourt J. Haguenau F: Problems raised by the search for virus particles in human leukemia. A study with the electron microscope of blood plasma, cerebrospinal fluid, and megakaryocytes from bone marrow. J Nat Cancer Inst 36:1089 1109, 1966 (10) Prosca AM, ADAMS WR: Virus-like particles in human plasma and serum: Role of platelet lysosomes. J Nat Cancer Inst 37:153 166, 1966 (11) DE HARVEN E, CLARKSON B, STUFE A: Electron microscopic study of human leukemic cells in tissue culture. Cancer 20:911–925, 1967 (12) de Harven E: Morphology of mumine lenkemia viruses. In Experimental Leukemia (Rich MA, ed.). New York, Appleton-Century-Crofts, 1998, pp 123 124 (13) NEWELL. GR, Harris WW, Bowman KO, et al: Evaluation of "virus-like" particles in the plasmas of 255 patients with leukemia and related diseases. New Eng J Med 278:1185 1191, 1968 (14) Ross A, Harnden D: Ultrastructural studies 011 normal and leukaemic human haematopoietic cells. Europ J Cancer 5:349–360, 1969 (15) DmochowWSKI L: Viruses and cancer in animals and man. Southern Med J 57:1267-1272, 1964 (16) STEWART SE, MICHIELI. EZ, Whang, JJ, et al: Viruses in human tumors. 1. Hodgkin's disease. J Nat Cancer Inst 43:1-14, 1969 (17) HALL, WT, MORTON DL, MALMGREN RA: Virus particles in tissue cultures of a human liposarcoma. J Nat Cancer Inst 44:507-513, 1970 (18) Moore DH: The milk agent. In Tumors Induced by Viruses: Ultrastructural Studies (Dalton AJ, Haguenau F, eds.). New York & London, Academic Press Inc., 1962, pp 113–150 (19) RUSTIGIAN R, JohnsION P, REHART 11: Infection of monkey kidney tissue cultures with virus-like agents. Proc Soc Exp Biol Med 88:8-16, 1955 (20) Clarke JK, Artridge JT, GAY FW: The morphogenesis of simian foamy agents. J Gen Virol 4:183188, 1969 JORNAL OF VIROLOGY, June 1971, p. 770-775 Copyright © 1971 American Society for Microbiology Vol. 7, No. 6 Printed in USA. Transformation of Murine Cells by Two "Slow Viruses," Visna Virus and Pro gressive Pneumonia Virus KENNETH K. TAKEMOTO AND LAWRENCE B. STONE Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014 Received for publication 24 February 1971 Visna and progressive pneumonia virus (PPV), two antigenically related, nononcogenic "slow viruses" which have ribonucleic acid (RNA)-dependent deoxyribonucleic acid (DNA) polymerase activity, were examined for their ability to transform cells. Murine cells which had been exposed to either visna or PPV developed foci of altered, spindle-shaped cells 3 to 4 weeks after infection. Visna and PPV transformed lines were established from these cultures. There was no evidence that other oncogenic DNA or RNA viruses were involved in the observed transformation. Visna or PPV could be "rescued" from all transformed lines by cocultivation with normal sheep testis cells. "Rescued" virus was identified as visna or PPV, and they retained the capacity to transform mouse cells. These experiments may have important implications in the understanding of both viral carcinogenesis and "slow" viral infections. Avian and murine ribonucleic acid (RNA)containing tumor viruses possess an RNA-dependent deoxyribonucleic acid (DNA) polymerase (1, 27). Since these original reports, many other C-type RNA viruses from other sources were also shown to contain this enzyme (5, 7, 16, 22), and it appears to be common to all oncogenic RNA viruses. On the other hand, of the nononcogenic RNA viruses thus far examined, only two, visna and the simian "foamy" virus (11, 14, 18, 23), have the virion-associated enzyme. Visna is the causative agent of a slow infection of the central nervous system of sheep (20, 21), whereas the "foamy" virus does not cause any known disease in monkeys. Progressive pneumonia virus (PPV), another "slow virus" which causes a slowly evolving lung disease of sheep (8), is a virus which has recently been shown to be antigenically related to visna (24) and also contains the RNA-dependent DNA polymerase(Stone and Takemoto, unpublished data). According to the hypothesis proposed by Temin (26), transformation by RNA tumor viruses involves a DNA intermediate which becomes integrated with the genetic material of the host cell. The viral polymerases are presumed to be involved in the synthesis of the DNA copy of the viral RNA. This hypothesis can be tested, at least in part, by the demonstration of oncogenic ac tivity by "nononcogenic" viruses which contain RNA-dependent DNA polymerase activity. We report here results of such experiments on the transforming capacity of visna and PPV in murine cells. MATERIALS AND METHODS Viruses and antisera. Visna virus and antiserum were obtained from H. Thormar (Institute for Basic Research, Staten Island. N.Y.). PPV was isolated from Montana sheep affected with the discase (8); detailed studies on PPV and its relationship to visna have recently been reported (24). Both viruses were grown and assayed by the plaque technique in a strain of sheep testis (ST) cells established in this laboratory and described in the previous study on PPV (24). Cell cultures and media. The AL/N mouse cell line was derived from mouse embryos of the AL/N strain; its properties have been described (25). The Balb-c cell line was also established in this laboratory from lungs of suckling Balb-c mice. Both cell lines exhibit a high degree of contact inhibition but like many mouse cell lines become "spontaneously” transformed upon prolonged passage (over 20 passages). In the experiments reported here, only carly passage cells (between 10 to 15 passages) were employed. Eagle's medium supplemented with 10% fetal bovine serum was used throughout. Serological tests. Complement fixation (CF) tests were done by the micromethod (18). For fluorescentantibody (FA) tests, the indirect procedure was used. 01. 7, 1971 VISNA AND PROGRESSIVE PNEUMONIA VIRUSES RESULTS Transformation of AL/N cells by visna and PPV, AL N cells were seeded at a concentration of 5 × 10 cells per 60-mm dish. The cultures were infected 24 hr later with an input multiplicity of 10 plaque forming units (PFU) per cell of visna or PPV. Growth medium was added after 2 hr of adsorption, and the cultures thereafter received fresh medium every 5 days. Cytopathic effects were never observed in cultures infected with either virus, and assay of supernatant fluids gave no evidence of viral replication. Approximately 3 weeks after virus exposure, morphologically altered, spindle-shaped cells made their appearance in the cultures infected with either visna or PPV. These were localized in discrete "foci" (Fig. 1A and B). The number of these foci was low, varying bteween 40 to 60 per dish. Approximately 10 PFU of visna or PPV were required per transformed focus. Cells from cultures showing foci as well as cells from uninoculated controls which did not contain altered cells were subcultured. Within two passages, the virus-infected cultures consisted entirely of uniform, spindle-shaped cells which formed a dense, multilayer of cells (Fig. 2A, B, and C). One line of visna- and two of PPV-transformed cells were established. In addition, cells from isolated foci of PPV-infected cultures were removed by the Puck cloning technique (15) and replated, and two clonal lines were derived. The established lines exhibited the following properties characteristic of cellular transformation: (i) altered morphology, (ii) rapid growth, and (iii) loss of contact inhibition of growth and movement (Fig. 2B and 2C). Upon continued passage, cells from two of the five lines lost their fibroblastic appearance and tended to become more epithelioid. The reason for this change is unknown. Virus "rescue." Supernatant fluids from transformed cells as well as 20% cell extracts were extensively tested for visna, PPV, or other agents by inoculation onto ST, mouse, and African green monkey kidney cells. Inoculated cultures were observed for 1 month and all tests were negative. When transformed cells at the third passage were tested for virus by co-cultivation with ST cells (10 transformed cells plated onto confluent ST cells), all lines yielded virus. In all cases, virus was not detected in the mixed cell cultures until 3 to 4 weeks. The rescued virus was identified as visna or PPV by distinct cytopathology (multinucleated giant cell formation) and by the FA technique with visna antiserum. The rescued virus was infectious for sheep cells only and failed to replicate in AL/N cells. 771 Infection of untransformed AL/N cells with rescued visna or PPV again resulted in cellular morphological alterations and transformed lines were established from these experiments. Failure to detect RNA or DNA tumor viruses or their antigens in transformed cells. The possibility that other oncogenic viruses may have been responsible for the transformation was considered, and experiments were conducted to rule this out. The presence of murine leukemia-sarcoma viruses or their antigens in the visna- or PPV-transformed cells was tested by several methods. (i) In CF tests, 20% cell extracts were tested against "broad-reactive" rat serum obtained from rats bearing transplantable Moloney sarcoma virus-induced tumors. (H. C. Turner, National Cancer Institute, kindly performed the CF tests for leukemia-sarcoma antigens.) The results of two different tests were negative. We have previously reported that highly purified visna virions do not have cross-reacting CF antigens with the murine Jeukemia-sarcoma viruses (23). (ii) Cells infected with and producing murine C-type RNA viruses induce the formation of syncytia when placed in intimate contact with XC cells, a line of rat cells transformed by Rous sarcoma virus (9, 17). This provides a sensitive method for detection and quantitation of virusproducing cells. Visna- or PPV-transformed cells at various concentrations up to 10' were mixed with 10 XC cells and plated onto 60-mm plastic dishes. The cultures were fixed and stained after 3 to 5 days and examined microscopically for areas of syncytial cells. All lines were negative in two different tests. It may be noted that visna or PPV did not have the capacity to cause cell fusion even when 10' PFU were inoculated onto confluent monolayers of XC cells. (iii) In the indirect FA tests, transformed cells were tested for leukemia-sarcoma antigens by using the same type of serum used in the CF test and fluorescein-conjugated goat antirat globulin. These were negative. Attempts to detect visna or PPV virus or viral antigens by the FA method with sheep antivisna or anti-PPV serum were also negative. The FA procedure was also used to rule out oncogenic DNA tumor viruses. Tests for the presence of virus-specific T antigens of simian virus 40, polyoma, and adenovirus 12 were done with hamster serum from animals bearing tumors induced by these viruses. All lines were negative for these antigens. Transformation of Balb-c cells with visna and PPV. Balb-c cells (15th passage) were exposed to visna and PPV under experimental conditions identical to those employed in the previous ex |