Chick Embryo Cell Cultures Research Articles

Optimization techniques: Studies in cell culture

AbstractWith the present large worldwide demand for vaccines and cellular by products, a number of anchorage-dependent cell mass culture systems have been developed. To obtain the maximum yield of product per run, the number of cells per system volume must be maximized and yet maintain efficiency. There is a large number of independent variables that could affect the cell yield. During the course of an experimental program to increase the cell yield, the usefulness of an existing computer optimization program was evaluated.A series of nine statistically designed experiments was done using multi-disk propagators for the mass culture of primary chick embryo cells. Cell input and time between medium refeeds were chosen as the independent variables from the many variables that could affect cell yield. The primary dependent variable was cell yield. The results indicated a substantial increase in cell yield as cell input was increased (57 to 85 percent increase by doubling the cell input) but a lesser increase ...

Induction and maintenance of 2',5'-oligoadenylate synthetase in interferon-treated chicken embryo cells.

Treatment of primary cultures of chicken embryo cells with homologous interferon results in a substantial increase in the level of 2',5'-oligoadenylate synthetase activity that can be detected in cell extracts. This increase can be prevented by inhibitors of RNA or protein synthesis and is thus thought to represent the induction of an interferon-inducible gene, perhaps the 2',5'-oligoadenylate synthetase gene itself. To examine this response in greater detail, we studied its kinetics under the following conditions: (i) cessation of interferon treatment after different lengths of time, (ii) delayed inhibition of RNA or protein synthesis, and (iii) combinations of these treatments. The results showed that in cells treated continuously with interferon, the enzyme level reached a peak after 9 h of treatment and then decreased with a half-life of about 30 h, despite the continued presence of interferon. Removal of interferon during induction reduced the peak level of activity that was attained and somewhat accelerated its decline but did not otherwise affect the time-course of the response. On the other hand, removal of interferon after maximum induction clearly accelerated the decay of enzyme activity. This process could be delayed by inhibitors of protein synthesis, which effectively stabilized the induced enzyme. This behavior is reminiscent of other inducible enzymes, such as the steroid-induced tyrosine aminotransferase, and suggests that the level of 2',5'-oligoadenylate synthetase, which is also inducible by steroid hormones in some cell types, is subject to similar control mechanisms.

Induction and maintenance of 2',5'-oligoadenylate synthetase in interferon-treated chicken embryo cells.

Treatment of primary cultures of chicken embryo cells with homologous interferon results in a substantial increase in the level of 2',5'-oligoadenylate synthetase activity that can be detected in cell extracts. This increase can be prevented by inhibitors of RNA or protein synthesis and is thus thought to represent the induction of an interferon-inducible gene, perhaps the 2',5'-oligoadenylate synthetase gene itself. To examine this response in greater detail, we studied its kinetics under the following conditions: (i) cessation of interferon treatment after different lengths of time, (ii) delayed inhibition of RNA or protein synthesis, and (iii) combinations of these treatments. The results showed that in cells treated continuously with interferon, the enzyme level reached a peak after 9 h of treatment and then decreased with a half-life of about 30 h, despite the continued presence of interferon. Removal of interferon during induction reduced the peak level of activity that was attained and somewhat accelerated its decline but did not otherwise affect the time-course of the response. On the other hand, removal of interferon after maximum induction clearly accelerated the decay of enzyme activity. This process could be delayed by inhibitors of protein synthesis, which effectively stabilized the induced enzyme. This behavior is reminiscent of other inducible enzymes, such as the steroid-induced tyrosine aminotransferase, and suggests that the level of 2',5'-oligoadenylate synthetase, which is also inducible by steroid hormones in some cell types, is subject to similar control mechanisms.

‘Red Book’ Update

UPDATE The Red Book Committee met on May 10, 1982 and considered a number of issues, including: 1. The 1982 edition (19th) of the Red Book has been distributed beginning on June 4, 1982. We welcome comments and suggestions for the 20th edition. 2. IMPORTANT REVISION IN RED BOOK RECOMMENDATIONS: After the Committee meeting it came to our attention that three children who had anaphylactoid reactions to egg ingestion experienced immediate allergic reactions to chick-embryo-grown live measles virus vaccine; two had difficulty breathing and one had hypotension. Persons who are egg-allergic but do not have a history of anaphylactoid reactions appear to be at little or no risk from live measles virus (LMV) vaccine (See Morbidity Mortality Weekly Rep 31:217-231, May 7, 1982). Because previous experience indicated no adverse reactions in egg-allergic children given vaccine prepared in chick embryo tissue culture, the 1982 Red Book contains the following statement: The vaccine currently used in the United States is prepared in chick embryo tissue culture by inoculation with a further attenuated passage of the Edmonston B strain of measles virus. This preparation is virtually devoid of allergenic substances derived from the chick embryo cell cultures used for growth of the live vaccine viruses. However, there is a remote potential risk of hypersensitivity reactions in patients allergic to eggs, chicken, or chicken feathers; large scale use of the vaccine for more than a decade has resulted in only rare, isolated reports of minor allergic reactions. In a study in which children known to be allergic to eggs, chicken, or chicken feathers were vaccinated with a chick embryo cell culture-derived vaccine, no allergic reactions were observed.

Newcastle disease virus stimulates the cellular accumulation of stress (heat shock) mRNAs and proteins

A biological agent, Newcastle disease virus, stimulated the synthesis of stress proteins in cultured chicken embryo cells. Previously, only physical and chemical agents were known to induce these proteins. The levels of translatable stress mRNAs were elevated in cells infected with avirulent or virulent strains; however, stress protein synthesis was stimulated strongly only in cells infected by avirulent strains. As did several other paramyxoviruses, avirulent strains of Newcastle disease virus stimulated the synthesis of glucose-regulated proteins as well as stress proteins. Possible stimuli of the synthesis of these two sets of proteins in paramyxovirus-infected cells are considered.

Effects of fowlpox virus infection on lipid metabolism in cultured chicken embryo cells.

Lipid biosynthesis was measured in cultured chicken embryo cells after infection with fowlpox virus. Between 24 and 72 h postinfection, fowlpox virus-infected cells incorporated less [14C]acetate and 3H2O into fatty acids and sterols than did mock-infected cells, demonstrating a virus-dependent inhibition of general lipid metabolism. Two specific effects of fowlpox virus infection were an accumulation of C-4 alkylated sterol intermediates and inhibition of monounsaturated fatty acid biosynthesis.

Ulcerative colitis and Crohn's disease tissue cytotoxins

Bowel-wall tissue filtrates from patients with inflammatory bowel disease produce cytopathic effects in tissue culture. The cytopathic effects inducers have been reported to have the characteristics of a small RNA virus. Clostridium difficile toxin also produces cytopathic effects and has been found in the stools of patients with Crohn's disease and ulcerative colitis. The present study concerns the further characterization of the cytopathic inducers in tissues of inflammatory bowel disease patients. It was found that they are nonsedimentable at 148,000 g for 2 h and resistant to inactivation by UV light. They are proteins that are distinct from C. difficile toxin and are unique cytotoxins which are associated with the early cytopathic effects observed in Rifffree chick embryo and rabbit ileum cell cultures. These results suggest that the early cytopathic effects previously described are not produced by a virus. They do not explain the delayed cytopathic effects seen in rabbit ileum or WI-38 cells.

Role of Methylation in the Induced and Spontaneous Expression of the Avian Endogenous Virusev-1: DNA Structure and Gene Products

The endogenous avian provirus ev-1 is widespread in white leghorn chickens. Although it has no major structural defects, ev-1 has not been associated with any phenotype and is ordinarily expressed at a very low level. In this report, we describe a chicken embryo (Number 1836) cell culture containing both ev-1 and ev-6 which spontaneously expressed the ev-1 provirus. This culture released a high level of noninfectious virions containing a full complement of virion structural (gag) proteins but devoid of reverse transcriptase activity or antigen. These virions contained 70S RNA closely related to the genome of Rous-associated virus type 0, but identifiable as the ev-1 genome by oligonucleotide mapping. A fraction of the RNA molecules in the 70S complex were unusual in that they were polyadenylated 100 to 200 nucleotides downstream of the usual polyadenylation site. Eight sibling embryo cultures did not share this unusual phenotype with 1836, indicating that it was not inherited. However, an identical phenotype was inducible in the sibling cultures by treatment with 5-azacytidine, an inhibitor of DNA methylation, and the induced expression was stable for more than 10 generations. Analysis of chromatin structure and DNA methylation of the ev-1 provirus in 1836 cells revealed the presence (in a fraction of the proviruses) of both DNase I hypersensitive sites in the long terminal repeats and in gag and a pattern of cleavage sites for methyl-sensitive restriction endonuclease not found in a nonexpressing sibling. These results lend strong support to the role of DNA methylation in the control of gene expression. Additionally, they explain the lack of phenotype associated with ev-1 as due to a combination of its low expression and defectiveness in pol and env.

Role of methylation in the induced and spontaneous expression of the avian endogenous virus ev-1: DNA structure and gene products.

The endogenous avian provirus ev-1 is widespread in white leghorn chickens. Although it has no major structural defects, ev-1 has not been associated with any phenotype and is ordinarily expressed at a very low level. In this report, we describe a chicken embryo (Number 1836) cell culture containing both ev-1 and ev-6 which spontaneously expressed the ev-1 provirus. This culture released a high level of noninfectious virions containing a full complement of virion structural (gag) proteins but devoid of reverse transcriptase activity or antigen. These virions contained 70S RNA closely related to the genome of Rous-associated virus type 0, but identifiable as the ev-1 genome by oligonucleotide mapping. A fraction of the RNA molecules in the 70S complex were unusual in that they were polyadenylated 100 to 200 nucleotides downstream of the usual polyadenylation site. Eight sibling embryo cultures did not share this unusual phenotype with 1836, indicating that it was not inherited. However, an identical phenotype was inducible in the sibling cultures by treatment with 5-azacytidine, an inhibitor of DNA methylation, and the induced expression was stable for more than 10 generations. Analysis of chromatin structure and DNA methylation of the ev-1 provirus in 1836 cells revealed the presence (in a fraction of the proviruses) of both DNase I hypersensitive sites in the long terminal repeats and in gag and a pattern of cleavage sites for methyl-sensitive restriction endonuclease not found in a nonexpressing sibling. These results lend strong support to the role of DNA methylation in the control of gene expression. Additionally, they explain the lack of phenotype associated with ev-1 as due to a combination of its low expression and defectiveness in pol and env.

Revertant analysis of a temperature-sensitive mutant of Newcastle disease virus with defective glycoproteins: implication of the fusion glycoprotein in cell killing and isolation of a neuraminidase-deficient hemagglutinating virus.

Biological and molecular properties of a temperature-sensitive mutant (C1) of Newcastle disease virus and its revertants were analyzed. C1 exhibited three temperature-sensitive alterations (plaque formation, virion assembly, and cytopathogenicity) and several defects which were also present at the permissive temperature. C1 virions contained low amounts of hemagglutinin-neuraminidase glycopeptides and consequently were deficient in hemagglutinating and neuraminidase activities. These virions also contained defective fusion glycoproteins which rendered them poorly hemolytic and slow to penetrate cultured chicken embryo cells. The biological activities of the membrane glycoproteins were recovered sequentially in a series of plaque-forming revertants. The coreversion of hemolysis, membrane-penetrating activities, and cytopathogenicity in the first-step revertant (S1) suggested that fusion glycoproteins were major contributors to cellular destruction. This revertant also provided evidence of a role for fusion glycoproteins in virion assembly. From S1 we isolated a large-plaque-forming revertant (L1) that assembled wild-type amounts of biologically active hemagglutinin-neuraminidase glycoproteins into virions. Although it was normal for hemagglutination, L1 had less than 3% of the neuraminidase activity of the wild type, demonstrating that these two activities can be uncoupled genetically. The neuraminidase deficiency of L1 did not impair its virulence in ovo or its reproduction in cultured cells.

Immunity to cytopathic agents associated with Crohn's disease: a negative study.

Serum and peripheral blood lymphocytes from 10 patients with Crohn's disease and 10 healthy subjects were examined for immunological reactivity against chick embryo cell cultures displaying cytopathic effects after inoculation with 0.2 micro filtrates prepared from Crohn's disease intestinal tissues. Although the assay systems (indirect immunofluorescence, lymphocyte transformation, and cytotoxicity) yielded positive results using well-characterized cytopathic viruses (mumps, measles), neither Crohn's disease nor healthy subjects showed immune reactivity to the chick embryo cell cultures inoculated with Crohn's disease intestinal tissues in any of the assay systems. These experiments provide evidence against the hypothesis that the in vitro cytopathic effect on chick embryo cell cultures produced by Crohn's disease intestinal filtrates are caused by a replicating virus or viruses.

Differential effect of hypertonic initiation block on the synthesis of collagen chains by cultured chick embryo cells.

The major type of collagen synthesized by fibroblasts or bone cells, type I collagen, consists of two chains normally found in a 2:1 ratio designated alpha 1(I)2 alpha 2(I) or more simply alpha 1(I)2 alpha 2. I have analyzed the relative synthesis of type I chains in these cells under conditions which reduce the initiation of protein synthesis. It was found that in bone cells, which make a large amount of collagen, the alpha 1(I):alpha 2 ratio is unaltered whereas in fibroblasts, which make smaller amounts of collagen, the alpha 2 chain is particularly sensitive to these same conditions. Examination of the collagen secreted into the medium, under these same conditions, also revealed an altered chain ratio from cells making low amounts of collagen.

Radio-sequence analysis of in Vivo multilabeled nonstructural protein ns86 of semliki forest virus

The N-terminal structure of in vivo multilabeled virus-specific enzymes and other proteins can be determined by single radio-sequence analyses. We now show this for Semliki Forest virus nonstructural protein ns86. The viral proteins were labeled with mixtures of up to seven different tritiated amino acids in cultures of chick embryo cells infected with the is-1 mutant. The multilabeled ns86 was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently degraded together with a carrier protein in a liquid phase sequencer. The resulting amino acid derivatives from each cycle of degradation were then separated by high-performance liquid chromatography and the radioactivity of each fraction was determined. The sequence obtained is sufficient to localize the nucleotide sequence on the 42 S RNA genome that codes for the N-terminus of ns86. Considerable differences were found in the incorporation of the labeled amino acids into ns86 during the in vivo conditions used. Stabilities and chromatographic separations of the amino acid derivatives vary and require extra precautions in radio-sequencing of multilabeled proteins.

Normal or Retrovirus-Infected Cultured Chicken Embryo Cells Express the 48,000 D Age-Related Antigen Characteristic of Embryonic Chicken Erythrocytes

The surface of normal or retrovirus-infected chick embryo cells was labelled with 125I using lactoperoxidase. The solubilized membrane material was allowed to react with antisera raised in rabbits to cultured chick embryo cells or to the membranes of embryonic or adult chicken erythrocytes. Analysis of the immunoprecipitates shows that chicken embryo cells not of erythropoietic origin express on their surface membrane an antigenic polypeptide of mol. wt. 48,000 daltons (D), which appears to be identical with that expressed on embryonic chicken erythrocytes.

Differential requirements for enriched atmospheric carbon dioxide content for intracellular growth in cell culture among selected members of the genus Rickettsia.

In an in vitro chicken embryo cell culture system, strains of Rickettsia prowazekii, R. mooseri, and R. rickettsii, but not of R. tsutsugamushi, required an atmosphere enriched in CO2 for intracellular growth.

53] Virus yield-reduction assays for interferon with the influenza virus neuraminidase assay

Publisher Summary This chapter describes virus yield-reduction assays for interferon with the influenza virus neurarninidase assay. The measure of reduction in yield of influenza A virus neuraminidase in infected cell cultures is a sensitive and reproducible bioassay for various interferons. A recombinant virus X7(F 1 ) (HON2) 3 is the most interferon sensitive of 12 influenza viruses tested and also produced higher yields of neuraminidase in most cell cultures than the other influenza viruses. This recombinant virus produces neuraminidase even in some cells that fail to produce infectious virus, but the best yields of neuraminidase were in primary chicken embryo cell cultures, which do produce infectious virus. The production of X7(F 1 ) neuraminidase is measured by the thiobarbituric acid method with the glycoprotein fetuin as substrate or by a method with the synthetic substrate methoxyphenylneuraminic acid.

A mouse model of the pathogenesis and postexposure prophylaxis of rabies.

A mouse model for the study of postexposure prophylaxis of rabies was established. Mice injected intramuscularly with a street strain of rabies virus were significantly protected from death by five daily 0.2-ml doses of inactivated rabies vaccine of chick embryo cell culture origin initiated immediately or 3 hr after infection. In these mice, a large amount of circulating interferon was induced as early as 1 hr after the first dose of vaccine and lasted until at least 12 hr but no such amount of interferon was induced by additional doses of vaccine. Serum antibody was first detected in the mice on day 6. It was noted that some of the surviving mice manifested an ataxia or paralysis of the legs. Increasing mortality rates were shown in mice treated with decreasing doses of the vaccine. Passive protection tests using concentrated IgG and IgM antibodies with equivalent neutralization titers showed that IgG antibody gave total protection when given 24 hr before the infection, while it was almost totally ineffective in reducing the mortality when given 2 days or more after infection. IgM antibody did not protect the mice even when given 24 hr before infection. These results suggest that interferon production is more important than antibody production in the initial stages of protection by postexposure vaccination. However, the mechanisms of postexposure prophylaxis in this model could not be explained only by the interferon produced by the vaccine and the possible contributions of additional mechanisms were suggested.

Viruses in Ixodes Uriae (Acari: Ixodidae) from Seabird Colonies at RøSt Islands, Lofoten, Norway1

A total of 1929 Ixodes uriae collected from Røst Islands, Lofoten, Norway, in July 1974, was divided into 204 pools and inoculated into suckling mice and chick embryo cell cultures for virus isolation. Virus was detected in 6.6% of the laboratory-molted female ticks, 5.4% of the males and 1.8% of the nymphs. No isolates were obtained from 149 unengorged adult ticks. Of 50 viral strains recovered, 30 belonged to the Uukuniemi group, 13 to the Kemerovo group, and I was a strain of Tyuleniy of Group B. Of 6 untyped strains, I was orbiviruslike and I resembled a coronavirus in a negative-staining electron microscopy. The infection rates of I. uria in Lofoten were similar to those reported in the Murmansk area in the northern USSR.

Two candidate G1 polypeptides in chick embryo fibroblasts.

AbstractResting cultures of primary chick embryo cells have been labeled with 3H‐leucine for the first 2 or 3 hours after feeding basal medium or basal medium supplemented with 10% dialyzed serum. The labeling patterns of the 3H‐polypeptides of the soluble cell fraction have been compared by fluorography of two‐dimensional gels. Large and consistent differences are seen in only three of the more than 900 spots that can visualized. This report concerns two of the three spots. The 3H contents of the two polypeptides (41,000 daltons, pI 7.1, designated 41–7.1, and 34,000 daltons, pI 6.2, designated 34–6.2) are increased by serum by about ten‐fold. The highly selective effect of serum on the labeling of the two spots does not appear to be an artifact related to the extractability, solubility, or state of aggregation of the polypeptides. The radio‐intensities of both polypeptides decrease markedly when the cells are labeled later than 3 hours after “shift‐up”.Drugs that inhibit RNA synthesis and are known to stop the progression of the chick cells through the G1 period, camptothecin, cordycepin and 5,6‐dichloro‐β‐D‐ribofuranosylbenzimidazole, depress, with great specificity, the enhanced labeling of polypeptide 41–7.1 in the stimulated cells, and all but camptothecin have a similar action on polypeptide 34–6.2. A high level of actinomycin D (10 μg/ml), but neither a low level of the drug (0.02 μg/ml) nor 5‐fluorouridine prevents the increased labeling of the two polypeptides in serum‐fed cells. That 5‐fluorouridine enters the chick cells and is converted to its active form is shown by the inhibition of the processing of pre‐ribosomal RNA.The observations with the RNA inhibitors are at least consistent with the conclusions that the enhanced labeling of the two sports results from increased rates of synthesis of the polypeptïdes that depend upon mRNA production but not on the formation of ribosomal RNAs, and that the polypeptides play a role in the regulation of DNA replication in the chick cells.

On the effect of inhibitors of mitochondrial macromolecular-synthesizing systems and respiration on the growth of cultured chick embryo cells.

We have found that chick embryo fibroblasts (DEF) cultivated in the presence of tryptose phosphate broth (TPB) are inherently resistant to the growth inhibitory effect of ethidium bromide (EB). As demonstrated by cytochrome oxidase activity and oxygen consumption measurements, analyses of reduced-minus-oxidized cytochrome spectra and electron microscopic observations, TPB did not seem to prevent the inhibitory effect of EB on mitochondrial DNA transcription. EB-treated chick cell populations cultivated in the presence of TPB behave essentially the same as populations treated with chloramphenicol (CAM) and grow with mitochondria devoid of a functional respiratory chain. In contrast to CAM-treated CEF populations, however, the respiratory activity of EB-treated cell populations did not reappear when the cells were shifted back to EB-free medium. Attempts to demonstrate that TPB confers resistance to the growth inhibitory effect of carbomycin and mikamycin, inhibitors of the mitochondrial protein-synthesizing system, have failed, the drugs being cytotoxic at doses where protein synthesis on mitoribosomes is not suppressed. On the other hand, the present results demonstrated that chick cell populations proliferate in the presence of the respiratory inhibitors rotenone, antimycin A, amytal and oligomycin whether or not TPB is present in the growth medium.