Type-C particles in human tissues. I. Electron microscopic study of embryonic tissues in vivo and in vitro

Thirty-two early-passage human embryonic cultures were examined in the electron microscope and one of them, derived from a muscle tissue, contained two budding and four mature type-C virus particles. Of the 15 original embryonic tissues from five embryos, only one showed the presence of a virus-like particle. It is of interest to note that the tissue specimens which contained virus-like particles originated from the same embryo.     

Aberrant viruses in cells infected with murine sarcoma virus-feline leukemia virus.

This study describes an unusual type of virus seen when a feline leukemia virus (FeLV) pseudotype of murine sarcoma virus (MuSV) obtained by cocentrifugation procedures infected feline embryo cells (FEF) and two Crandell cat cell lines (CrFK1, CrFK2). When all three cell cultures were infected with MuSV-FeLV, only FEF and CrFK2 were transformed and only these showed normal and aberrant virus. The CrFK1 infected with MuSV-FeLV did not transform but did replicate normal type-C virus with a 50-A intermediate coat. The virus replicated in the two transformed lines showed three particles; a normal particle with a 50-A intermediate coat, a normal particle with a 100-A intermediate coat, and an aberrant particle with a 100-A intermediate coat.     

Murine leukemia virus-associated cell surface antigens in rats neonatally infected with Gross murine leukemia virus.

The inoculation of newborn W/F, Lew, AS and DA rats with Gross murine leukemia virus (G-MuLV) resulted in the prompt appearance of cells with viral protein antigens (VPA) on their surfaces. These were first found in the bone marrow and spleen and later in the thymus gland. As the animals developed, the VPA-positive population expanded and the intensity of the fluorescence increased. In the spleen, the cells with the strongest fluorescence had the properties of T-cells, but in both spleen and bone marrow low levels of VPA were found on non-T-cells. The VPA-positive population expanded long before malignant cells could be detected and, in most animals, the entire T-cell compartment became antigen-positive. These animals were unable to respond to G-MuLV antigens and many eventually developed leukemia. However, some animals apparently broke the tolerance that followed neonatal infection and eliminated VPA-positive cells from their tissues     

Spontaneous and induced appearance of murine leukemia virus antigen containing cells in organ cultures of embryonic mouse thymus.

The potential of embryonic thymus organ cultures for studies on relations of endogenous MuLV, lymphoid cells and thymic microenvironment to lymphoma development were evaluated. Four main findings are reported. First, thymuses of 14-day-old CBA and AKR embryos could be maintained in organ cultures for at least 9 weeks with sustained production of lymphocytes. Lymphopoiesis in CBA and AKR thymuses were not grossly different. Secondly, an indirect immunofluorescence (IF) technique demonstrated spontaneous appearance of MuLV-antigen-containing cells in AKR, but not in CBA thymuses. Such spontaneous MuLV expression first occurred after 16 days of organ culture, thereafter infrequently and at random in individual thymus cultures. Thirdly, incubation of AKR and CBA thymuses in lymphoma extract containing AKR-type MuLV at initiation of organ cultures induced MuLV-antigen-containing cells. These were first detected after 7-14 days in culture, somewhat earlier and initially more frequently in AKR than in CBA thymuses. In the former, induction was accompanied by a clear reduction in the number of lymphocytes per thymus. Fourthly, iododeoxyuridine (IdUrd) treatment of AKR thymuses on cultute day 0, 3 or 7 decreased the number of lymphocytes per thymus and induced appearance of MuLV-antigen containing cells, assayed 8-20 days later. The IdUrd effect was most marked on day 0, and decreased sucessively on days 3 and 7. IdUrd had a much slighter effect on CBA thymuses, causing a lower reduction in cell numbers and inducing few MuLV-antigen cells. These main results clearly demonstrate the potential usefulness of the organ culture system for studied on leukemogenesis. It may be directly applied to answer several questions raised by detailed findings in our study.     

Isolation of a precipitating glycoprotein antigen from cell cultures persistently infected with bovine leukemia virus.

A procedure was developed to isolate a glycoprotein with precipitating antigen activity from fluids from fetal lamb kidney cell cultures persistently infected with bovine leukemia virus (BLV). The antigen was precipitated by ammonium sulfate and subjected to affinity chromatography on concanavalin A Sepharose. The glycoprotein was eluted with alpha-methyl-D-mannoside and was further purified by gel filtration over Sephadex G-100. Antigen activity was determined by agar gel immunodiffusion (AGID) reactions with serum from cattle infected with the virus. The major portion of the AGID activity was eluted from the Sephadex G-100 in the 60,000-dalton elution region. In some experiments, identical AGID activity was also found in the 18,000-dalton elution region. The larger protein was discovered to have a molecular weight of 58,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its designation as a glycoprotein was confirmed by carbohydrate-positive staining. The isolated BLV glycoprotein antigen did not contain ovine or bovine proteins as indicated by gel immunodiffusion.     

Electron microscopy study of virus particles in reproductive organs of mice inoculated with leukemia virus (Moloney)

Ultra-thin sections of various reproductive organs of BALB/c mice inoculated with Moloney leukemia virus were examined in the electron microscope. Various tissues such as testis, seminal vesicles, prostate, vas deferens, vas efferens, epididymis and ovary revealed the presence of a considerable number of virus particles similar in appearance to those observed in spleen, thymus and bone marrow of leukemic mice. A large number of virus particles were seen budding into the ducts of vas deferens and vas efferens. These morphological studies may point to the possibility that virus produced by infected cells of the male reproductive organs is released into the semen and under suitable circumstances may be transmitted to normal females at coitus through seminal fluid.     

Electron microscopic studies of intracisternal virus particles in Soehner-Dmochowski murine sarcoma virus-induced bone tumors of New Zealand Black rats.

Soehner-Dmochowski murine sarcoma virus (Moloney)-induced bone tumors of New Zealand Black rats carry two morphologically different types of virus particles, namely, extracellular type C and intracisternal virus particles, which have thus far not been reported. These two types of virus particles have also been observed in the tissue culture cells derived from normal prostate tissues of A/Dm and BALB/c/Dm mice after inoculation of cell-free extracts of these bone tumors. The intracisternal virus particles, 90 to 120 nm in diameter, have always been found in the rough endoplasmic reticulum; they have two inner concentric layers with a relatively electron-lucent center, frequently showing cylindrical, chain-like, or multipolar budding forms. Type C virus particles produced by Soehner-Dmochowski murine sarcoma virus (Moloney)-infected prostate tissue culture cells from A/Dm and BALB/c/Dm mice belong to the murine sarcoma-murine leukemia virus group, as revealed by the fixed immunofluorescence test and by immunoelectron microscopy. The morphological and immunological relationship of intracisternal virus particles and other types of virus particles (such as type C, type H, and intracisternal type A virus particles) and intracisternal virus particles in guinea pig leukemia are defined by routine electron microscopy observations and by immunoelectron microscopy studies.     

Virus expression in different tissues of normal and tumor-bearing mice inoculated with a murine leukemia virus.

Evolution of virus expression in different lymphoid organs as well as in solid syngeneic tumors of mice inoculated with an MuLV was studied with the aid of in vitro XC co-culture technique. When normal adult mice of strain XLII were inoculated intraperitoneally with a cultured Rauscher virus (RC), the virus could be detected, 10 days after inoculation, only in bone marrow in small amounts and thereafter no virus could be found in any of the organs tested, including bone marrow, spleen, thymus, lymph node and kidney. However, when age- and sex-matched parallel mice bearing syngeneic subcutaneous non-viral tumors were inoculated similarly with the RC virus, the virus could be detected abundantly not only in bone marrow and spleen but also in tumors during the first 3 weeks and even 6 weeks after virus inoculation. Transitional decrease or disappearance of the virus was observed around the 25th-31st day in organs and tumors of the inoculated mice. When the tumor mass was removed from these mice by surgery, the virus disappeared rapidly and definitely from all the organs tested. The virus recovered from in vitro explanted and cultured tumors, taken from mice inoculated with the virus, induced typical lymphoid leukemia in BALB/c mice inoculated as newborns. However, from certain aspects (hypertrophy of the thymus and lymph nodes), this virus was different from the original RC virus.     

Interactions of murine leukemia virus (MuLV) with isolated lymphocytes. I. Virus replication in lymphocytes infected with Friend virus and cultures in diffusion chambers in vivo.

A new technique for infection of mature lymphocytes with murine leukemia virus (Friend) MuLV-F) is described. Spleen cells for normal, non-infected donors were placed into diffusion chambers (constructed with 0.22 mum por size Millipore filters) which were then implanted into the peritoneal cavities of normal syngeneic recipient mice. The cells were infected with an injection of MuLV-F into the peritoneal cavity and, in some instances, also by placing virus into the chambers. Cells were recovered by treating the chamber content with elastase and collagenase. The infection was determined in two ways: (1) cells with replicating MuLV were enumerated as infection centers (IC) on S+L- indicator cells; and (2) virus-related cell membrane antigen (MA) was detected by immunofluorescence. Cells recovered from chambers after 2-3 weeks of culture represented about 10% of the original inoculum; viability was approximately 90%. The number of IC in MuLV-F-infected chambers was about 10 times higher than obtained by infection and cultivation of spleen cells in vitro. The kinetics of IC and MA in chamber-cultured. MuLV-F-infected spleen cells was similar to that in the spleen of infected mice during the first 10 days after infection. Later on, the process of infection within the chambers slowed down, reaching about 50% MA-positive and about 10% IC-positive cells, whereas the number of both IC- and MA-positive cells in the spleen reached 80% or more. The infection of splenic lymphocytes in diffusion chambers occurred equally well when chambers were implanted into: (1) syngeneic, virus susceptible hosts; (2) syngeneic, lethally irradiated hosts; and (3) allogeneic, virus-resistant hosts, suggesting that the process within the chamber is independent of MuLV replication in the tissues of the chamber-bearing mouse. The diffusion chamber technique seems to provide an environment in which various types of isolated lymphocytes of different mouse strains can interact with MuLV almost as efficiently as in vivo.