Transformation-enhancing factor(s) produced by virus-transformed and established cells.

Chick embryo fibroblasts (CEF) and hamster BHK21 cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) release into the culture medium a factor or factors which enhance 2- to 7-fold the formation of transformed foci by chich embryo fibroblasts infected with the Bryan strain of RSV (B-RSV). The factor(s) also increase the number of foci failing to revert to normal phenotype at restrictive temperature (41 degrees C) in cultures infected with a temperature-sensitive mutant (FU-19) of SR-RSV which is defective for transformation. The factor(s) is produced also by BHK21 cells transformed by other tumor viruses and by BHK21 cells passaged for a long time, but not by normal CEF, CEF transformed by B-RSV, CEF infected by FU-19 at 41 degrees C, normal hamster embryo fibroblasts, established but density-inhibited mouse fibroblasts, or BHK21 cells of early passages. The relative enhancement of the number of B-RSV foci can be more than 100-fold when the medium contains fetal calf serum which suppresses focus formation in controls. The focus-enhacing factor(s) appears to act after infection and has been termed, operationally, transformation-enhancing factor(s) or TEF. The factor produced by RS2/3 cells which enhances the formation of B-RSV foci is non-dialyzable and thermolabile, and is presumably a protein. Its molecular weight is between 10(5) and 2 X 10(5) daltons.     

Lack of correlation between Marena tumor induction and expression of endogenous avian RNA tumor virus genome.

Infection with Marek's disease virus (MDV) and subsequent tumor development failed to induce the expression of endogenous avian RNA tumor virus genome in Line-15I and Line-7 chickens which lacked such expression at 1 day of age. Titers of avian leukosis virus (ALV) group-specific (gs) antigen detected by the COFAL test and expression of chick helper factor (chf) activity remained relatively constant in birds which expressed these genome functions at hatching time. Endogenous ALV belonging to the E-subgroup was isolated from two of 50 birds; both were controls not exposed to MDV. There was no correlation between expression of ALV genome (gs or chf) at 1 day of age and the rate of tumor development subsequent ot MDV infection. It was concluded that MDV infection can induce tumors without the participation of endogenous ALV genome.     

Myeloid leukaemogenicity and transmission of the HPRS-103 strain of avian leukosis virus.

The HPRS-103 strain of avian leukosis virus (ALV) was isolated recently from meat-type chickens and represents a new envelope subgroup. Its oncogenicity has been studied in three meat-type and five Leghorn strains of chickens. In the meat-type strains, the virus, following embryonal inoculation, induced an overall incidence of 27% myelocytic myeloid leukosis (myelocytomatosis) and 12% renal adenomas, with long median latent periods. Amongst the Leghorn lines, these tumors occurred with similar incidence in line 0, but with lower or zero incidences in the other lines. A variety of other tumours occurred with low incidence. Embryonal infection resulted in a permanently tolerant viraemic state with shedding of ALV group specific (gs)-antigen to egg albumen; contact infection resulted mainly in the development of non-shedder birds with serum virus-neutralising antibodies. Contact infection in a meat-type line was associated with the development of transient or permanent viraemia in some birds, and a low tumour incidence. A viraemic phase was not detected following contact infection in a Leghorn line and no tumours developed. The long latent period between embryo infection and tumour mortality, apparently differing from the consequences of infection with acutely transforming ALVs, and the inability of HPRS-103 ALV to transform cultured bone marrow cells, suggests that this virus may lack a viral oncogene and exert its oncogenic properties by some other mechanism such as promoter insertion activation of a cellular oncogene.     

Extractable herpesvirus antigens from chickens with Marek's disease

Marek's disease herpesvirus (MDHV) antigens are demonstrable by indirect immunofluorescence and micro-immunodiffusion tests in epithelial cells of feather follicles (FF) of chickens infected with GA strain of MDHV; but lymphoid tumor cells are devoid of virus particles and virus-associated antigens. The antigens extracted from virus-containing epithelial cells growing in vivo or in vitro were found to be of a similar nature. Electron microscopy showed that antibody-positive chicken sera caused specific coating of MDHV particles extracted from feather follicular cells.     

Avian retrovirus-induced surface antigens and their cross-reactivity with chemically-transformed cells and primary embryonic cells of Japanese quails.

By testing spleen cells from avian leukosis (ALV) and avian sarcoma virus (ASV)-injected Japanese quails in a microcytotoxicity assay against various target cells, we have demonstrated the existence of several target antigens. With non-transformed ALV-infected Japanese quail cells used as target cells, an avian retrovirus subgroup-specific destruction was obtained when spleen cells from animals infected with either avaian sarcoma or leukosis virus of the same subgroup were employed. This reaction is probably due to the virus envelope glycoproteins (Ve-gp) expressed on the cell surface. Apart from this subgroup-specific reaction, avian retrovirus group-specific destruction of ASV-transformed cells was demonstrated by means of effector cells immunized with ASV of a different subgroup. This reaction is restricted to transformed cells and not due to the virus envelope glycoprotein because the same effector cells are not cytotoxic to ALV-infected non-transformed cells but cytotoxic to sarcoma-virus-transformed cells which lack Ve-gp. Quail methylcholanthrene-tumor cells which show a transformation phenotype similar to that of ASV-transformed cells but which are free of detectable endogenous and exogenous retrovirus were also destroyed by the spleen cells from ASV tumorbearing animals. The same effector cells also exerted a weak cytotoxic effect on uninfected primary embryo cells but not to embryo cells after several passages.     

Cellular and humoral anti-tumor immune responsiveness in chickens bearing tumors induced by avian sarcoma virus.

A systematic comparison was undertaken of the respective abilities of normal chicken embryo fibroblast (CEF) cells, Rous sarcoma virus (RSV)- transformed CEF cells, avian Rous sarcoma (RS) tumor cells and murine RS cells to serve as targets and antigen donors in various assays for the detection of cellular and humoral anti-tumor immunity in chickens bearing tumors induced by Rous sarcoma virus. As measured by a cytotoxicity procedure, avian and murine RS cells were more susceptible to the killing effects of sensitized lymphocytes than were transformed CEF which in turn were more reactive than normal CEF. In contrast, sera from tumor-bearing animals were able to stain by indirect immunofluorescence only the avian RS and transformed CEF cell types. Extracts of both transformed CEF cells and avian RS cells but not normal CEF were equally effective as inhibitors of migration of peritoneal exudate cells derived from tumor-bearing animals. Transformed CEF were found to produce far higher quantities of transforming virus progeny than avian RS cells, although the latter were apparently albe to synthesize defective viral particles. These data indicate the significant variations which may occur under experimental conditions, depending on the types of assays and target cells employed.     

Transplantable primate tumors induced by Rous sarcoma virus. I. Induction of tumors transplantable into young marmosets

The tumors induced in white-lipped marmosets (Saguinus fuscicollis, S. nigricollis) by Rous sarcoma virus (RSV) of chicken origin (RSV-SR) were not transplantable to allogeneic hosts. In contrast, RSV rescued from these tumors (RSV-M) induced sarcomas that were transplantable to young but not to adult marmosets. The tumors induced by RSV-M and the transplants rapidly enlarged, metastasized to various organs, and killed the recipients 29-59 days post inoculation. Cell lines were readily established from all transplantable sarcomas. No virus expression was detected in transplantable tumor cell lines by electron microscopy or by biochemical and biological assays. However, RSV of the same subgroup as RSV-SR was rescued from both short-term and long-term tumor cell cultures by cocultivation with chicken embryo fibroblasts (CEF). The rescued viruses transformed marmoset cells 100-fold more efficiently than CEF cells, although CEF cells remained permissive for virus replication. Cytogenetic studies revealed extensive chromosome abnormalities in tumor transplants but not in RSV-M-induced sarcomas. All cell lines were hyperploid and contained structurally abnormal, large metacentric and telocentric chromosomes. Immunologic studies failed to detect group-specific (gs) antigen of the avian sarcoma-leukemia complex in either RSV-M-induced, transformed cells or tumor transplants. By complement-dependent cytotoxicity assays, with the use of marmoset anti-gs serum, RSV-associated antigen could be detected on the surfaces of tumor cells. No differences in the expression of this antigen existed between transplantable and nontransplantable marmoset sarcomas. All transplantable cell lines contained abnormal amounts of lipids and glycogen in comparison to RSV-SR-induced tumors and normal marmoset cell lines. The glycogen was associated with unique cytoplasmic membrane complexes and was surrounded by either single- or double-membraned vesicles.     

Wide host range of murine sarcoma virus

Cultures of canine, rabbit, porcine, avian, feline, bovine, simian and human origin were tested for their susceptibility to the Kirsten murine sarcoma virus (Ki-MSV). Data indicated that Ki-MSV replicates and transforms the cultures of canine embryo, rabbit kidney, porcine kidney, feline embryo, bovine embryo and human tumor cells. These morphologically altered cells contained infectious virus and group-specific (gs) complement-fixing (CF) antigen characteristic of viruses of the murine sarcoma-leukemia complex. The infected cultures produced type-C virus particles. RNA-dependent DNA polymerase activity was also demonstrable in the altered cells. The present results indicate that members of the murine sarcoma-leukemia virus complex, particularly Ki-MSV, exhibit a wider host range than was hitherto believed.     

Lymphomas resembling lymphoid leukosis in chickens inoculated with reticuloendotheliosis virus.

Chickens inoculated as embyros or at hatching with the chick syncytial strain of reticuloendotheliosis virus developed a high incidence of lymphoid neoplasms between the 17th and 43rd weeks of age, involving principally the liver and bursa of Fabricius. On the basis of organ distribution, latent period, pathology and surface IgM production, the lymphomas closely resembled those of lymphoid leukosis. One inoculated chicken developed a myxosarcoma. No tumors were observed in uninoculated controls. The tumor-bearing chickens were free of infection with Marek's disease virus and exogenous avian leukosis virus (ALV) of subgroups A, B, C or D. However, the chickens were known to express endogenous ALV genes to varying degrees.     

A variant Schmidt-Ruppin strain of Rous sarcoma virus with increased affinity for mammalian cells

SR-RSV-D(H), a variant virus with extremely high tropism for mammalian cells, was isolated by passage of the Schmidt-Ruppin strain of Rous sarcoma virus of subgroup D (SR-RSV-D) through hamster cells. This variant virus has acquired an altered envelope glycoprotein, encoded by the env gene, that has high affinity for receptors on the surface of mammalian cells. The variant virus transforms rat cells at about 100 times the efficiency of the parental virus, SR-RSV-D(S), as assayed by focus formation. Addition of amphotericin B (Fungizone) to the medium at a concentration of 0.2 micrograms/ml completely inhibited rat cell transformation by SR-RSV-D(H), possibly by blocking virus penetration into the cells, whereas the drug showed no inhibitory effect on transformation of chick embryo fibroblast (CEF) cells by the variant virus or on transformation of rat cells by the parental virus. The efficiency of transformation of rat cells by the variant virus was much less than its efficiency of transformation of CEF cells. Analysis of infection of rat cells suggested that the virus can infect rat cells as efficiently as CEF cells but that rat cells were not transformed by the virus as fully as CEF cells because of inefficiency of some post-penetrational step involved in viral gene expression. The finding that E1AY cells, rat cells expressing adenovirus E1A gene, were transformed by SR-RSV-D(H) as efficiently as CEF cells supports this conclusion and suggests that expression of the E1A gene in rat cells may overcome the defect in the transforming step(s) in rat cells.     

Antibody response to related leukosis viruses induced in chickens tolerant to an avian leukosis virus.

White Leghorn chickens congenitally infected with a subgroup A avian leukosis virus ALV-F42 were challenged at various ages with the serologically related subgroup A avian leukosis viruses (ALV) RAV-1, RAV-3, and RAV-5 and their antibody responses and viremic status determined. Birds given injections of RAV-1 failed to produce neutralizing antibody to any of the pseudotype sarcoma viruses of the various ALV, whereas many birds challenged with RAV-3 or RAV-5 produced neutralizing antibody directed against the heterologous RAV-1 as well as the homologous viruses, but not against the tolerated virus ALV-F42. Virtually all birds remained viremic despite the presence of neutralizing antibody. The results suggested that a phenomenon akin to circumvention or abrogation of tolerance to ALV-F42 was effected, and that there are multiple determinants of the viral envelope antigens of ALV as measured by neutralization.     

Expression of Thy 1 antigen in normal and neoplastic mammary cells of mice.

The expression of Thy 1.2 (theta C3H) antigen was measured on the membranes of normal and neoplastic RIII and C3H mammary cells. Competitive inhibition assays revealed that the average membrane content of Thy 1.2 in mammary tissues was about equal to that of lymph node cells. Higher percentages of Thy 1.2-positive cells than mammary tumor virus (MuMTV)-positive cells were observed by immunofluorescence, which suggested that not all the Thy 1.2-positive cells recovered from tumors were also MuMTV-positive. Established tissue culture cell lines C3H and RIII MT expressed lower levels of Thy 1.2 than did cells from mammary tumors. Treatment with the synthetic gluco-corticoid dexamethasone increased the average Thy 1.2 expression in cultured mammary tumor cells as well as the levels of RNA-directed DNA polymerase. Since the percentages of Thy 1.2-positive cells also were greater in steroid-treated cultures, while fewer cells were needed to absorb a standard amount of anti-Thy 1.2 activity, it was concluded that dexamethasone enhanced membrane Thy 1.2 expression as well as MuMTV production by the cultured cells.     

Infectious DNA recovered from avian tumor-virus-producing cells.

A single treatment of chick embryo fibroblasts with DNA recovered from chick embryo fibroblasts productively infected and transformed with four different strains of RSV, or productively infected with two different strains of RAV, resulted in virus production and cell transformation (in the case of RSV) two or three passages after treatment (8-25 days). The virus recovered from cultures was phenotypically identical to that produced by the donor cells. No virus production nor cell transformation resulted from treatment of control cultures with DNA digested with DNAse. Infectious RSV-DNA was recovered from purified donor cell nuclei and was associated with the precipitable fraction of DNA prepared according to the method of Hirt (1967). It also sedimented with cellular DNA in density gradients, and with high molecular weight DNA (2-4 times 10-7 daltons) in sucrose gradients, which suggests that it is associated and may be integrated with chromosomal DNA. In some experiments, DNA fractions of lower molecular weight (down to 6 times 10-6 daltons) were also infectious. DNA from virus-producing RSV-transformed cells also gave rise to virus and Rous cells in cultures of fibroblasts from gs- embryos. However, the amount of DNA required for successful infection varied widely between experiments, and no reproducible dose-effect relationship was observed. The frequency of DNA-treated cells which produced virus remained low, even when the assay cultures were pretreated with 5-bromodeoxyuridine.     

Rous sarcoma recurrence and Rous sarcoma virus growth in chicken muscle

Following Rous sarcoma virus (RSV-Bryan standard strain) infection and tumor regression in chickens, tumors recurred in 3 of 6 chickens after contralateral inoculation of 10⁵ of RSV. In an attempt to understand the mechanism of tumor recurrence, chickens which had recovered from RSV infection were inoculated at the regression site with Rous associated virus (RAV). Tumors recurred in one-third of those chickens that received three bi-weekly RAV injections and in one chicken that received only a single RAV injection. The tumors re-appeared from 6 to 19 weeks after regression of the original tumors and within 4 weeks of the last RAV challenge. RSV was recovered from cultures of a recurring tumor as well as from a wing that developed subcutaneous blistering following RAV inoculations. RSV was also obtained from 40% of “recovered” wing muscles explanted in tissue culture up to 3 months after regression. In addition, RSV was isolated from a tumor that recurred in one of 12 recovered chickens inoculated with Freund's adjuvant. In an attempt to determine if ageing of muscle contributed to resistance to infection and suppression of tumor recurrence, wings of 5-week-old and 12-week-old chickens were infected with RSV and RAV, or RAV alone. RSV and RAV were recovered from most muscles explanted 48 h or more after RSV infection of 5-week-old chickens. However, when muscles from 12-week-old chickens were explanted 4 days after infection, very little RSV was recovered, although most of the RAV-infected wings produced RAV in culture. This suggests that RAV growth in older muscles is probably not the limiting factor in RSV growth. The relationship between various factors involved in resistance to tumor development and tumor recurrence in chickens recovering from RSV infections is discussed.     

Oncogenesis by Marek's disease herpesvirus in chickens lacking expression of endogenous (gs, chick helper factor, Rous-associated virus-O) and exogenous avian RNA tumor viruses.

Chickens free of exogenous avian leukosis virus (ALV) infection, replicating endogenous ALV (Rous-associated virus-O), gs antigen, and chick helper factor were fully susceptible to induction of Marek's disease (MD) by ALV-free MD viruses. Dual infection with Rous-associated virus-2 and MD virus did not significantly alter the character of the MD lesions. Thus exogenous ALV infection was not requisite for MD virus-induced oncogenesis. Although participation of endogenous RNA tumor virus genes in MD lesion induction could not be excluded, expression of such genes in MD tumors as gs antigen was not established.     

Pathogenesis of Marek's disease: early appearance of Marek's disease tumor-associated surface antigen in infected chickens.

Marek's disease tumor-associated surface antigen (MATSA) appeared in different lymphoid tissues of P-line chickens soon after they were infected with BC-1 strain of Marek's disease virus. MATSA-bearing cells first appeared in spleens by 5 days post infection (PI) and were observed through a 21-day experimental period at mean levels varying from 3.8 to 21.9% of the total cells examined. Lower percentages of MATSA-bearing cells were observed in the thymus, in the bursa of Fabricius, among peripheral blood lymphocytes, and among bone marrow cells beginning 7 days PI. The antigen was not detected on normal lymphocytes from chickens or chicken embryos, nor was it detected on cultured chicken embryo fibroblasts infected or transformed by avian RNA tumor viruses.     

Role of 3′-end of viral genome in tumor heteroinduction by the avian sarcoma virus

Transformation defective virus was derived by restriction endonuclease cleavage from a clone of the avian sarcoma virus Schmidt-Ruppin strain, strongly oncogenic for rats. The transfection experiments of chicken cells by digested proviral DNA gave rise to transformation defective virus. The td virus was possible to recover in vivo in chickens. The tumors obtained after a long latent period contained the sarcoma virus which was able to transform chicken cells in vitro and to induce tumors in chickens. All viruses, parental, td- and recovered were of D subgroup specificity. The tumor induction experiments in rats have shown that the recovery of viral genome deletion in td mutant by cellular sequences was not enough to regain the oncogenicity for rats. The results stressed the importance of 3-end sequences of the virus genome, probably the sequences in C region for heteroinduction ability of the avian sarcoma virus.