ISOLATION OF A SARCOMA VIRUS FROM A SPONTANEOUS CHICKEN TUMOR Shigeyoshi ITOHARA, Kouichi HIRATA, Makoto INOUE, Masanori Veterinary Pathology, Faculty of Agriculture, Yamaguchi University* HATSUOKA, and Akio SATO A spontaneous tumor was obtained from a hen of White Leghorn stock raised in a local farm in Yamaguchi City, in December 1973. The tumor had characteristics of fibro-or myxofibro-sarcoma and could be maintained by transplantation in chickens of similar flocks. This tumor was proved to produce sarcoma virus at 33rd passage and the transforming virus was designated as Y73 sarcoma virus. Biological studies suggested that the virus recovered from the tumor belonged to avian leukosis-sarcoma complex having subgroup A specificity. The possibility of defectiveness of this virus will be discussed. Rous12) reported the isolation of sarcoma virus from a transplantable chicken sarcoma in 1911 and since then Fujinami sarcoma virus6) and Bratislava 77 of avian sarcoma virus (B77)15) had been isolated from similar chicken tumors of different origin. Only these three strains and thier variants have been used as representative strains of avian sarcoma virus. They produce sarcomas in chickens at inoculated sites and induce transformed foci in chick embryo fibroblast cell cultures.8) Bryan strain of Rous sarcoma virus (RSV) is defective in replicating capacity,7) while other strains of RSV and B77 are non-defective.4) In addition, each strain of RSV and B77 is also different in their characters to alter cellular adenylate cyclase activity of transformed cells.19) Therefore, the addition of newly isolated virus to avian sarcoma virus group will be helpful for further analysis of this virus group. Materials and Methods Viruses: Avian leukosis viruses used were RAV-1 of subgroup A and RAV-60 of subgroup E. The sarcoma viruses were Schmidt-Ruppin strain of Rous sarcoma virus subgroup A and D (SR-RSV- A and SR-RSV-D), Prague strain of Rous sarcoma virus subgroup A (PR-RSV-A), B77 of subgroup C, and pseudotypes of Bryan high-titer strain of Rous sarcoma virus belonging to subgroups A, B, C, and E [BH-RSV (RAV-1), BH- RSV (RAV-2), BH-RSV (RAV-7) and BH-RSV (RAV-60)]. Y73 Virus: The original stock was prepared by the method described by Moloney10) from a solid tumor of 3-week-old chickens induced by inoculation with tumor cells maintained by serial transplantation in chickens for 3.5 years. The starting material for tissue culture experiment was obtained from the medium of monolayer cultures of C/O (gs-, chf-) cells heavily infected with the original stock. Cells: Chick embryo fibroblast (CEF) and quail embryo fibroblast (QEF) cultures were prepared from 10-to 11-day-old chicks and quail embryos obtained from the Kannonji Institute of the Research Foundation for Microbial Diseases, Osaka University, Kannonji, Kagawa-ken. C/O and C/BE CEF used in this study were gs-negative ones. The tests for chf were performed as described previously by Weiss et al.18) Cell culture and focus assay were followed by the standard procedures.17) For enhancement of virus adsorption, polybrene was added to the medium at a final concentration than subgroup A virus.16) Other conditions of cell cultures were described by Owada et al.11)
S. ITOHARA, ET AL. Interference Test: Viral interference test was carried out by the procedure described by Rubin and Vogt.14) Test for Virus Production of Foci: CEF cultures (106 cells/60-mm dish) were infected with serially diluted virus and overlaid with agar medium 18 hr later. Seven to 10 days after infection, single foci from the cultures which contained 5 foci or less were isolated separately onto CEF feeder layers in 15-mm wells. When confluent they were transferred to 35-mm dishes. The supernatants of these cultures were harvested at 6 to 10 days after focus isolation. After three cycles of freezing and thawing, they were tested in C/O CEF for the presence of transforming virus. Infectious Center Assay: Procedure described by Weiss et al.18) was adopted for infectious center assay. Pock Formation (of Sarcoma Virus): Virus was inoculated onto the chorioallantoic membrane (CAM) of 10-day-old embryos through an artificial air space and pocks formed on CAM were scored 5 days later. Observation with Electron Microscopy: The bits of tumor were fixed with 4% gultaraldehyde in 0.1M phosphate buffer and then with 1% OsO4 in the same buffer. They were dehydrated with ethanol and embedded in Epon mixture. Thin sections were stained with uranyl acetate and lead citrate, and observed with an electron microscope, Histachi HS-8. Results and Discussion Isolation of a Sarcoma Virus from a Spontaneous Transplantable Tumor: A spontaneous tumor, found in a hen of White Leghorn stock raised in a local farm in Yamaguchi City in December 1973, had characteristics of fibro- or myxofibro-sarcoma (Photo 1). The tumor had been maintained by transplantation in chicks of a flock over several years. In order to isolate a transforming, filtrable agent, the tumor was homogenized and the cell-free extract was prepared. Original stock of the virus was first tested for tumorigenicity. Three-week-old chickens were inoculated with 0.1ml of the stock in the wing web. Tumors were recognized at the inoculated sites about 8 days after inoculation. Under an electron microscope, virus-like particles were observed in extracellular space as well as in vacuoles of tumor cells. The particles contained an electron-dense inner core surrounded by less dense material enveloped with double limiting membranes (Photo 2). These particles were morphologically similar to type-c viruses reported by Bernhard.1) Pocks were formed when the original stock was inoculated on CAM of 10-day-old embryos. The pocks resulted from a focal proliferation of the epithelium followed by a limited infiltration of the sarcoma cells into the underlaying mesoderm (Photo 3). These observations were in accordance with those previously described by Rubin.13) Transformation of CEF and QEF was observed after inoculation with the original virus stock. Transformed cells in cultures were more refractive than normal cells, and assume round or spindle forms. Further, transformed cells lost the parallel orientation of normal fibroblasts, and had an increased Table I. Host Range of Y73 and Enhancement with Polybrene Table II. Interference of Avian Leukosis Viruses with Y73 Gann
tendency to pile up on each other (Photo 4). This transforming virus was tentatively designated as Yamaguchi 73 sarcoma virus (Y73). Host Range of Y73: Table I shows the focus-forming ability of Y73 on two types of CEF, C/O (susceptible to all subgroups), C/BE (resistant to subgroups B and E), and on QEF. QEF is fully susceptible to viruses of subgroups A and E,4) and partially susceptible to subgroup C viruses.5) Efficient transformation was observed on C/O and C/BE CEF as well as QEF. These results suggested that this virus has the host range of subgroup A. However, the enhancement of plating efficiency of Y73 with polybrene reserved the possibility that the virus stock was a mixture of subgroup C and subgroup E. Interference Patterns of Y73: Y73 was interfered with RAV-1 but not with RAV-60 (Table II). This result indicated that Y73 has the characteristics of subgroup A, and subgroup E virus should be a minor component if present. In order to define the envelope character of Y73, the virus was cloned successively three times by isolating a single focus from LSOLATION OF A NEW AVIAN SARCOMA VIRUS (FFU). After the final cloning, Y73 was assayed on C/O, C/BE, and C/BE CEF infected with RAV-1. As shown in Table III, the virus infected C/BE as well as C/O CEF and was interfered with RAV-1. This indicated that cloned Y73 had the same envelope properties of subgroup A as uncloned virus. Replicating Capacity of Y73: Presence of a 100-fold excess of non-transforming virus of subgroup A was demonstrated in the original stock of Y73 by the interference test after end-point dilution. The association of subgroup A non-transforming virus might occur during transplantation of tumors in conventional chikens. Therefore, we tried to isolate pure Y73 by cloning. Table IV shows the frequency of non-producing foci at each cloning. Comparatively high-ratio of non-producer clones was obtained at 1st and 5th cloning. On the other hand, even after the 4th cloning of virus-producer foci, large excess of associated virus was found in the cultures when transformed cells became predominant. Since these results suggested that Y73 may be a defective virus, replicating capacity was tested by the infectious center assay. As shown in Table V, focus-forming capacity of clone 1 was reduced to about one-tenth of the control when the infected cells were killed with mitomycin-c and overlaid with uninfected cells. The same treatment caused essentially no reduction in Table III. Host Range of Y73 Cloned Three Times (Clone No. 10) Table IV. Virus Production of Foci Induced by Y73 Table V. Infectious Center Assay
focus-forming capacity of PR-RSV-A which is known to be non-defective ASV. The result of infectious center assay supported the defectiveness of Y73. However, the maintenance of non-producer transformant was difficult and most transformed cells were lost after several transfers. Therefore, we could not define the defectiveness of Y73. Further study is needed to determine whether or not Y73 is defective in replicating capacity. In addition to three representative strains of ASV, several spontaneous chicken tumors producing ASV-like agents had been isolated2,3,9) but unfortunately these strains were lost without characterization. We described the isolation and some characterization of a sarcoma virus from a transplantable chicken tumor of spontaneous origin. Before and during the period of transplantation in chickens, no avian sarcoma virus had been used either in our laboratory or in the local chicken farm indicating that the agent is a new isolate of avian sarcoma virus. The authors are indebted to Prof. Kumao Toyoshima, Osaka University, for valuable advice and encouragement, and to Dr. Masatoshi Owada, Osaka University, for helpful advice and supply of known viruses. The authors are also grateful to the Research Foundation for Microbial Diseases of Osaka University for generous supply of fertile eggs. REFERENCES (Received May 15, 1978) S. ITOHARA, ET AL. Photo 3. Histopathological finding of the pock. Photo 4. Focus of transformed cells induced in EXPLANATION OF PLATES Photo 1. Histopathological finding of the transplantable tumor. Hematoxylin and Eosin. Photo 2. Electron micrograph of C-type virus (a) By Y73. (b) By BH-RSV (RAV-1). Gann
ISOLATION OF A NEW AVIAN SARCOMA VIRUS
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