Transformation of chick embryo fibroblasts by reticuloendotheliosis virus.

A strain of reticuloendotheliosis virus-infected chicken embryo fibroblasts has been isolated and found to be transformed by the following criteria: (1) altered morphology, (2) increased cell density, (3) increased passage capability, (4) growth in soft agar, (5) increased uptake of 2-deoxy-D-glucose, and (6) tumorigenicity. Detectable avian leukosis-sarcoma viruses are not produced by these cells.     

Actin organization in chick embryo fibroblasts after influenza virus infection. I. Isolation and characterization of actin from chick embryo cells

Comparison of two starting materials for actin purification has shown that preparation of actin from aceton-dried cytoskeleton was more effective than from native chick embryos (CE). The isolated actin formed a single band of Mr = 42-43000 in SDS-PAGE; less purified samples revealed additional faint bands. G form of actin (non-polymerized) inhibited the activity of DNase I, electron microscopy showed actin filaments and bundles formed upon its polymerization. The freshly purified homogeneous actin has not lost its DNase I-inhibiting activity when incubated for 60 min at 35 degrees or 45 degrees C. Older or less purified actin samples kept under similar conditions showed 18-25% decrease of their DNase I-inhibiting activity and a loss of their polymerization ability. Digestion with trypsin caused a decrease of DNase I-inhibiting activity of fresh as well as for older actin samples.     

Replication of poxvirus DNA in chick embryo fibroblasts

Replication of vaccinia virus DNA in chick embryo fibroblasts at a high multiplicity of infection started after 4-6 hr, reached a maximum at the 12th hr and was completed 18 hr after inoculation (p.i.). An analysis of newly synthesised DNAs in the period of the highest rate of synthesis revealed the formation of short (10-30S) fragments which within 30 min were transformed into single-stranded molecules with a sedimentation coefficient of 62S. The final stage of closing of the ends of single-stranded molecules needed much more time and the bulk of "mature" single-stranded 90 S DNA molecules was observed 17 hr p.i.     

Age-related changes induced by tunicamycin in wheat germ agglutinin binding to chick embryo fibroblasts

The specific roles of N-acetylglucosaminyl and sialyl residues were investigated in the binding of wheat germ agglutinin (WGA) to fibroblasts from 8- and 16-day chick embryos. Cells from the 8-day embryos exhibited two classes of WGA binding site, whereas fibroblasts from 16-day embryos only displayed one. Neuraminidase treatment of fibroblasts from 8-day embryos raised the number of WGA binding sites with a high affinity constant and reduced the number of sites with a low affinity constant. In 16-day cells, neuraminidase treatment reduced the number of WGA binding sites. The data presented here suggest that WGA binds to cell-surface glycoproteins containing sialic acid residues. This tendency was more marked in 16-day than in 8-day cells and corresponded to the finding that neuraminidase released more sialic acid from the surface of 16-day cells than of 8-day cells. Glycosylation of cell-surface N-linked glycoproteins did not seem essential in determining the WGA binding capacity, as shown by the effect of tunicamycin on fibroblasts from both 8- and 16-day embryos. In the absence of N-glycosylated binding sites, WGA bound to O-glycosylated structures and the older tunicamycin-treated embryo cells exhibited a larger number of WGA binding sites than the younger tunicamycin-treated cells, in relation to the increase of the amount of O-glycosylated structures during embryo development.     

Alterations in membrane polypeptides of chick embryo fibroblasts induced by transformation with avian sarcoma viruses.

Membrane proteins of chick embryo fibroblasts (CEF) transformed with various strains of avian sarcoma viruses were analyzed by electrophoresis in SDS-polyacrylamide gels and compared with those of untransformed cells. The following differences were consistently detected in CEF transformed with B77, the Prague strain of Rous sarcoma virus (PR-RSV) or the Schmidt-Ruppin strain of RSV (SR-RSV): (1) The appearance of a polypeptide band with an apparent molecular weight of 90,000, (2) increase in amount of a polypeptide of 79,000 daltons, (3) significant decrease in amount of a polypeptide of 50,000 daltons and (4) marked decrease in amount of a protein of 200,000 daltons. CEF infected with the temperature-sensitive (ts) mutants of these strains, LA334 (of B77), LA31 (of PR-RSV) or OS122 (of SR-RSV) showed similar changes at 36°, but at 41°, except for alteration (4), the profiles of the membrane proteins were similar to those of uninfected cells. Changes (1) and (3) were reversible and clearly observable within a few hours after a temperature shift of CEF infected with ts mutants. Fusiform transformation induced by a variant of B77 was also shown to induce alterations (1) and (3).From these and other results, the appearance of the polypeptide band of 90,000 daltons, which could not be detected in untransformed cells, and the marked decrease in amount of a protein of 50,000 daltons in cell membranes were concluded to be closely correlated with transformation of CEF.     

Polypeptides induced in chick embryo cells by Kemerovo virus

Fifteen polypeptides induced by Kemerovo virus were detected in chick embryo cells (Mr 140, 98, 89, 72, 65, 62, 57, 54, 50, 47, 43, 41, 39, 31 kD, and 30 kD). Nine of them, namely the 140, 98, 65, 62, 57, 54, 50, 47 kD, and 41 kD polypeptides were also found in the partially purified virus. However, the latter contained also considerable amount of host cell proteins, predominantly the 205 kD, 45 kD, and 37 kD polypeptides. In the electron microscope the spherical viral particles exhibited a poorly defined surface structure of a diameter of 70-75 nm.     

Integration and differentiation of human embryonic stem cells transplanted to the chick embryo.

Human embryonic stem (ES) cells are pluripotent cells that can differentiate into a large array of cell types and, thus, hold promise for advancing our understanding of human embryology and for contributing to transplantation medicine. In this study, differentiation of human ES cells was examined in vivo by in ovo transplantation to organogenesis-stage embryos. Colonies of human ES cells were grafted into or in place of epithelial-stage somites of chick embryos of 1.5 to 2 days of development. The grafted human ES cells survived in the chick host and were identified by vital staining with carboxyfluorescein diacetate or use of a green fluorescent protein-expressing cells. Histologic analysis showed that human ES cells are easily distinguished from host cells by their larger, more intensely staining nuclei. Some grafted cells differentiated en masse into epithelia, whereas others migrated and mingled with host tissues, including the dorsal root ganglion. Colonies grafted directly adjacent to the host neural tube produced primarily structures with the morphology and molecular characteristics of neural rosettes. These structures contain differentiated neurons as shown by beta-3-tubulin and neurofilament expression in axons and cell bodies. Axons derived from the grafted cells penetrate the host nervous system, and host axons enter the structures derived from the graft. Our results show that human ES cells transplanted in ovo survive, divide, differentiate, and integrate with host tissues and that the host embryonic environment may modulate their differentiation. The chick embryo, therefore, may serve as an accessible and unique experimental system for the study of in vivo development of human ES cells.     

Viral polyproteins in chick embryo fibroblasts infected with avian sarcoma leukosis viruses.

The soluble RNA-dependent RNA polymerase activities in the 105,000-g supernatant fraction of homogenates prepared from uninfected and tobacco mosaic virus (TMV)-infected tobacco leaves have been compared. The enzymes were purified 40- to 50-fold from both mock-inoculated and TMV-infected leaves and were found to have identical behavior upon (NH₄)₂SO₄ fractionation, Sephadex G-100 gel filtration, and DEAE-Bio-Gel and phosphocellulose chromatography. Although no qualitative differences in polymerase activity were detected, the specific activity of the polymerase from infected leaves was greater than that from mock-inoculated leaves at each step in the purification. Moreover, the most highly purified enzymes from both types of leaf tissue were indistinguishable with respect to a number of catalytic parameters, viz., requirements for RNA synthesis, kinetics of RNA synthesis, lack of template specificity, and character of the RNA products such as strandedness, T_m, size, and complementarity to the TMV-RNA used as the template. SDS-polyacrylamide gel electrophoresis of the proteins in the most highly purified enzymes showed that both preparations contained 2 major and at least 13 minor corresponding polypeptides; none were unique to TMV infection. The evidence in toto suggests that the enhanced soluble RNA-dependent RNA polymerase activity following TMV infection is due to a stimulation of a host RNA-dependent RNA polymerase rather than to the genesis of a new viral-coded RNA polymerase. The possible relationships between the soluble host polymerase, the membrane-associated TMV replicase, and a 130,000-MW viral-coded polypeptide thought to be involved in TMV-RNA replication remain to be resolved.     

Glycolipids of chick embryo fibroblasts infected with temperature-sensitive mutants of avian sarcoma viruses.

The effect of virus-induced transformation on the glycolipid pattern of chick embryo fibroblasts has been studied with mutants of avian sarcoma viruses which are temperature sensitive for the initiation and maintenance of transformation. The transformed state is associated with a three- to fivefold decrease in the level of hematoside. This decrease is temperature dependent with the mutants, and at the nonpermissive temperature normal cell morphology and behavior as well as normal hematoside levels are restored. The higher sialosyl glycolipids of chick embryo fibroblasts also decrease in transformation, but in mutant-infected cells at the nonpermissive temperature they do not return to normal levels.     

Interferon pretreatment primes interferon production by human adenovirus in chick embryo cells.

Pretreatment of chick embryo cells (CEC) with homologous interferon enhanced the subsequent interferon production induced by human adenovirus. This priming effect of interferon pretreatment was also demonstrable when trypsin-treated virus was used for the induction of interferon.     

The ultrastructure of cultures of chick embryo fibroblasts containing Togavirus-like particles

The ultrastructural appearance of fibroblasts cultured from specific pathogen-free chick embryos is described. These cells contained virus-like particles approximately 50 nm in diameter which accumulated in cytoplasmic vacuoles. In their size, morphology and mode of replication these particles closely resemble viruses in the genus Pestivirus, family Togaviridae.     

Interaction of interferon with chick embryo cells

summary The degree of resistance to vesicular stomatitis virus (VSV) increased with the time of contact of chick embryo cells with interferon at 37°C. Resistance developed in cells which were held in contact with interferon at 4°C for an hour, washed free of interferon and then incubated at 37°C before inoculation. The degree of resistance that developed after such treatment was lower than that formed when both the contact with interferon as well as further incubation were performed at 37°C. Increased time of contact with interferon at 4°C led to increased protection. This is considered evidence that the resistance developed after treatment at 4°C was not due to residual unbound interferon carried over to 37°C. Instead it must have been the result of some kind of interaction between interferon and cells taking place at 4°C. Incubation of cells with interferon in the presence of dimethylsulfoxide (DMSO) not only failed to increase, but even significantly decreased, the degree of resistance. Incubation of cells with a high concentration of fetal calf serum (FCS) inhibited the action of interferon and increased the yield of VSV.Medical Student, supported by the Honors Program of New York University School of Medicine, maintained by Public Health Service Grant 5T5 GM2-05.     

Productive influenza virus infection of synchronized chick embryo fibroblast cells.

The effects of cell metabolic activity on the outcome of influenza virus infection were studied in partially synchronized chick embryo fibroblast cultures. There was no evidence to show that the time in the cell cycle at which cells were infected had any significant effect on the final virus yield. However, some differences were detected in the length of the latent period between infections established in synchronized or in stationary cells. Influenza virus could replicate in synchronized or normal cell cultures in which DNA synthesis was inhibited with 9-beta-D-arabinofuranosyladenine (ara-A).     

Passage of herpes simplex virus type 1 on chick embryo fibroblasts confers virulence for chick embryos

The pathogenesis of chorioallantoic membrane (CAM) infection with herpes simplex virus 1 and 2 (HSV-1 and HSV-2) as well as chick embryo fibroblast (CEF) passaged HSV-1 was studied. It was found that HSV-2 is at least a million-fold more virulent than HSV-1 as measured by pfu/LD50 ratios for the embryo. Serial passage of HSV-1 in vitro on CEF cells selected for a virus (CEFP10) which, unlike its parental HSV-1 strain, is able to kill the embryo. The restriction endonuclease maps of CEFP10 and its parental strain are indistinguishable and the mechanism of the increased virulence of CEFP10 was demonstrated to be its enhanced replication in chick embryo cells both in vivo and in vitro. In contrast, despite its inferior replicative ability, HSV-2 was found to have a biologically important specific invasiveness function that is not simply related to overall viral replication. Finally, the ability to isolate HSV-1 (CEFP10) virulent for the chick embryo after passage in vitro illustrates that tissue culture passage of HSV in appropriate cells may actually increase virulence for the animal host.     

Morphology of A/WSN influenza virus-infected chick embryo cells.

A/WSN (H0N1) influenza virus-infected chick embryo cells (CEC) attained a swollen central region 4 hours post infection (p.i.), different from the elongated uninfected cell. From 6 hours p.i. of CEC an increasing number of approximately 100-120 nm, round protrusions were observed by scanning and transmission electron microscopy on the cell membrane, which was smooth on the uninfected cell. At 48 hours p.i. a great number of protrusions were observed on the cell; in addition, there were a number of holes or invaginations in the membranes of the infected cells indicating cell destruction.