Chicken Embryo Fibroblast Cultures Research Articles

Phenotypic Analysis of Yellow Fever Virus Derived from Complementary DNA

A thorough phenotypic characterization of yellow fever (YF) virus generated from cDNA is a necessary prerequisite for mapping virulence/attenuation determinants and exploring the potential of YF attenuated virus 17D as a carrier for heterologous protective epitopes. In this study, YF virus was produced from 17D cDNA clones after lipofectin-mediated RNA transfection of certified primary cultures of chicken embryo fibroblasts (YFiv5.2/SL). This virus was passaged once in embryonated chicken eggs according to current YF vaccine manufacture methodology to produce the experimental virus (YFiv5.2/VL). These viruses were characterized in established monkey neurovirulence safety tests and quantitative clinical and histologic scores were derived for each virus. The experimental vaccine viruses (YFiv5.2/SL and VL) compared favorably with another well-known YF vaccine strain (17DD) used as control virus for the histologic score. Although slightly higher clinical neurovirulence was observed for YFiv5.2 as compared with the 17DD virus, it should not preclude the use of YFiv5.2 for mapping YF virus virulence determinants.

Allergic Reactions to MMR Vaccines in Egg- and Non-Egg-Sensitive Children: A Continuing Controversy

Measles vaccines prepared in chick embryo fibroblast culture have been used since 1963. Since then, dramatic reductions of the incidence of measles have been observed where the vaccination was extensively applied in the pediatric population, with the best results observed in the initial phase when the target population was that producing over 90% of cases in the prevaccinal era. Since the virus vaccine is grown in cultures of chicken fibroblast cell, although egg proteins could not be found in vaccines, a possible limiting factor to the diffusion of measles vaccination is represented by the assumption that allergy to egg proteins contraindicates the vaccination. It is known that there is a controversy in the literature about the safety of measles vaccine and measles, mumps, and rubella vaccine (MMR) in eggallergic children. Recent data suggest that reactions to MMR vaccine can be mediated by gelatin and/or neomycin. In this paper, current knowledge on the safety of measles immunization in children with and without egg allergy is discussed. In addition, we propose that all children to be immunized with MMR vaccine and their families should be questioned about neomycin and related antibiotic allergy.

Cleavability of hemagglutinin from an extremely virulent strain of avian influenza virus containing a unique cleavage site sequence.

An avian influenza virus, A/turkey/England/50-92/91 (H5N1), showed extremely high virulence in chickens, although its hemagglutinin (HA) cleavage site sequence (R-K-R-K-T-R), having a nonbasic (Thr) residue at the second position (P-2) from the carboxyl terminus of HA1, does not conform to the previously established consensus sequence motif, X-X-R/K-X-R/K-R (X = nonbasic residue), for highly virulent phenotype of the H5 virus. When we evaluated the HA cleavability of this strain in chicken embryo fibroblast culture, we observed that, unlike other HAs with a Thr residue at P-2, this HA was efficiently cleaved. These findings suggest that a nonbasic residue at the P-2 does not affect its recognition and catalyzation by cleavage enzymes that are otherwise influenced by steric structure around the cleavage site.

The relocalization of v-Rel from the nucleus to the cytoplasm coincides with induction of expression of Ikba and nfkb1 and stabilization of I kappa B-alpha

The v-Rel oncogene induces the expression of major histocompatibility complex class I and II proteins and the interleukin-2 receptor more efficiently than does c-Rel (R. Hrdlicková, J. Nehyba, and E. H. Humphries, J. Virol. 68:308-319, 1994). The kinetics with which these immunoregulatory receptors are induced in B- and T-lymphoid cell lines and chicken embryo fibroblast cultures expressing c-Rel or v-Rel have been examined. v-Rel induced the expression of major histocompatibility complex classes I and II and interleukin-2 receptor more efficiently than did c-Rel at later times after infection. In all three cell types, this increased efficiency was accompanied by a shift in the majority of v-Rel from the nucleus of the cytoplasm. The concomitant relocalization of v-Rel was also demonstrated during the in vitro transformation of spleen cells. The translocation coincided with increased steady-state levels of I kappa B-alpha. Coninfection by retroviral vectors expressing v-Rel, I kappa B-alpha, or NF-kappa B1 demonstrated that either I kappa B-alpha can contribute to the shift of v-Rel to the cytoplasmic compartment. The induction of nfkb1 and Ikba mRNA and the stabilization of I kappa B-alpha by v-Rel were shown to be responsible for these effects. In comparison with c-Rel, the expression of v-Rel was associated with lower levels of transcription of these genes. However, the ability of v-Rel to stabilize I kappa B-alpha remained unchanged. The ability of v-Rel to stabilize I kappa B-alpha but poorly induce Ikba mRNA expression relative to c-Rel may play a role in regulating gene expression, thereby leading to transformation.

Native TIMP-free 70 kDa progelatinase (MMP-2) secreted at elevated levels by RSV transformed fibroblasts.

Rous sarcoma virus-transformed cultures of chicken embryo fibroblasts (RSVCEF) secrete elevated levels of a 70 kDa progelatinase, an avian form of the 72 kDa matrix metalloproteinase-2 (MMP-2). Affinity-purified preparations of secreted 70 kDa progelatinase are composed of two distinct populations of zymogen: a 70 kDa progelatinase tightly complexed with an avian form of TIMP-2 and a native 70 kDa progelatinase free of any detectable TIMP-2. These two forms of the progelatinase can be separated by Mono Q FPLC in the absence of denaturing agents. The homogeneity of the two separated forms is demonstrated by both SDS-PAGE and nondenaturing, native gel electrophoresis. The purified TIMP-free 70 kDa progelatinase is stable in aqueous conditions and does not spontaneously autoactivate. Treatment of the TIMP-free progelatinase with the organomercurial, p-aminophenylmercuric acetate (APMA), results in rapid (5-60 minutes) autolytic conversion of the 70 kDa progelatinase to 67 kDa, 62 kDa and lower molecular weight forms of the enzyme. APMA treatment of the TIMP-free progelatinase yields a preparation that is enzymatically active with a high specific activity towards a peptide substrate. Identical treatment of TIMP-complexed progelatinase with APMA results in a significantly slower conversion process in which the 70 kDa progelatinase is only 50% converted after 6-24 hours and the specific enzyme activity of the preparation is 8 to 18-fold lower. Purified avian TIMP-2 added to the TIMP-free progelatinase forms a complex with the progelatinase and prevents the rapid autolytic conversion induced by APMA. Comparative analysis of parallel cultures of transformed RSVCEF and normal CEF demonstrates that the transformed cultures contain threefold higher levels of the TIMP-free progelatinase than the normal CEF cultures which produce predominantly TIMP-complexed progelatinase. The presence in transformed cultures of elevated levels of a more readily activated TIMP-free progelatinase, the suppression of its rapid activation by TIMP-2, and the potential effect of the altered balance between TIMP-free and TIMP-complexed 70 kDa progelatinase on the invasive, malignant phenotype, are discussed.

Partial Inhibition by Turkey Herpesvirus of Serotype 2 Marek's Disease Virus Plaque Formation and in vivo Infectivity

The increased use of serotype 2 Marek's disease virus (MDV) and serotype 3 turkey herpesvirus (HVT) as components of effective bivalent vaccines against Marek's disease (MD) prompted studies on the possible interactions of these two viruses in vitro and in vivo. The replication of the SB-1 strain of MDV was compared with replication of the FC126/2 strain of HVT in chickens and cell cultures infected with one or both viruses. Replication of MDV was reduced in the presence of HVT in both in vitro and in vivo systems. MDV plaque counts in dually infected chicken embryo fibroblast cultures inoculated with tissue-culture-propagated viruses were reduced by up to 91%; however, no inhibition was noted when inocula consisted of virus-infected buffy-coat cells. Plaque formation by MDV in chicken embryo fibroblast cultures was inhibited by virus-free conditioned medium from HVT-infected cultures. This conditioned medium also inhibited growth of vesicular stomatitis virus in a standard interferon assay. In chickens inoculated with both MDV and HVT, MDV viremia titers were lower and the dose required to infect 50% of susceptible chickens was increased 13-fold compared with chickens inoculated with MDV alone. In spite of these findings, there was no evidence that high concentrations of HVT interfered with either the ability of MDV to induce protective synergism in vivo or the protective efficacy of bivalent vaccines. No reciprocal inhibitory effects of MDV on the replication of HVT in vivo or in vitro were noted.

The effects on cellular functions of bile acid and trypsin in stagnant bile juice in anomalous arrangement of the pancreaticobiliary duct

To examine the reasons for the high frequency of biliary tract carcinogenesis in individuals with anomalous arrangement of the pancreaticobiliary duct (AAPBD), we investigated the effects on cellular functions of bile acid and trypsin, which are possible risk factors for carcinogenesis found in stagnant bile juice, using a chick embryo fibroblast culture system. Bile acid was found to increase PGE 2 synthesis which has been shown to be increased in premalignant lesions, but to suppress the incorporation of [ 3H]-labelled TdR into DNA. On the other hand, trypsin increased the incorporation of [ 3H]TdR into DNA, but did not increase PGE 2 synthesis. These results suggest that both the bile acid and trypsin present in the stagnant bile juice in AAPBD act to stimulate cell proliferation, but that their mechanisms of action on cell growth differ. Therefore, the combination of these effects of different types of tumor promoters in the stagnant bile juice in AAPBD may account for the high incidence of carcinogenesis.

A base-paired structure in the avian sarcoma virus 5' leader is required for efficient encapsidation of RNA.

Selective encapsidation of avian sarcoma-leukosis virus genomic RNA within virions requires recognition of a cis-acting signal (termed psi) located in the 5' leader of the RNA between the primer binding site and the splice donor site. Computer analyses indicate the potential for numerous secondary structure interactions within this region, including alternative conformations with similar free energy levels. We have constructed mutations designed to disrupt and restore potential secondary structure interactions within psi to investigate the role of these structures in RNA packaging. To test for the ability of psi mutants to package a heterologous reporter gene into virions, chimeric constructs bearing avian sarcoma virus 5' sequences fused to lacZ were transiently cotransfected with a nonpackageable helper construct into chicken embryo fibroblasts. lacZ virions produced from cotransfected cells were used to infect new cultures of chicken embryo fibroblasts, and then an in situ assay for individual cells expressing lacZ was done. Results obtained with this assay were confirmed in direct analyses of isolated virion RNA by RNase protection assays. Two mutations, predicted to disrupt a potential stem structure forming between elements located at nucleotides 160 to 167 and 227 to 234, severely inhibited packaging when either element was mutated. A construct in which these mutations were combined to restore potential base pairing between the two elements displayed a partially restored packaging phenotype. These results strongly suggest that the structure, referred to as the O3 stem, is required for efficient encapsidation of avian sarcoma virus RNA. Site-directed mutagenesis of additional sequence elements located in the O3 loop reduced packaging as measured by the indirect assay, suggesting that these sequences may also be components of the encapsidation signal. The possible implications of the O3 stem structure with regard to translation of avian sarcoma-leukosis virus short upstream open reading frames are discussed.

Development of an in vitro cell culture model to investigate the induction and quantification of oxidative stress and its inhibition by α-tocopherol

The ability of the natural antioxidant α-tocopherol to protect against oxidative stress in vitro was assessed. Primary cultures of chicken embryo fibroblasts (CEF) were oxidatively stressed by exposure to paraquat (PQ). Activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were measured as indices of oxidative stress. CEF incubated with 0.125–1.0 m m PQ for 18 hr exhibited increased SOD activity ( P < 0.05). CAT activity increased with 0.25 m m PQ ( P < 0.05). GSH-Px activity decreased significantly in the presence of PQ. No cytotoxicity, as indicated by lactate dehydrogenase release, was observed at PQ concentrations below 2 m m. Incorporation of added α-tocopherol (100 n m) into 0.25 m m PQ-treated CEF resulted in SOD activity not significantly different from that observed in control cells not treated with PQ. Lower levels of added α-tocopherol (16 n m) returned CAT to its control value. However, even at 1000 n m α-tocopherol, GSH-Px activity was not protected in PQ-treated cells. CEF represent a useful model to study both inducers and inhibitors of oxidative stress.

Mutations in the nsP1 Coding Sequence of Sindbis Virus Which Restrict Viral Replication in Secondary Cultures of Chick Embryo Fibroblasts Prepared from Aged Primary Cultures

SVMPA, a mutant of Sindbis virus (SV), which is able to replicate in Aedes albopictus cells treated with mycophenolic acid (MPA) or ribavirin, also has a host range phenotype. This phenotype is most clearly demonstrated by means of efficiency of plaquing (EOP) assays on secondary chick embryo fibroblasts (CEF) prepared from aged primary CEF. For example, in one experiment in which standard SV (SVSTD) had a relative EOP (EOP on primary CEF ÷ EOP on secondary CEF) of 1.6 the corresponding value for SVMPA was 2340. The host restriction of SVMPA was also seen with similarly prepared secondary cultures of duck embryo fibroblasts, but not with established lines of quail cells. The finding that the accumulation of viral RNA was much lower in SVMPA-infected secondary CEF than in SVSTD-infected CEF indicated that the replication of SVMPA in these cultures was blocked at an early step. Revertants of SVMPA were isolated which were no longer host-restricted but had retained their resistance to MPA. Of the three mutations [nucleotide (nt) 120, 127, and 963] in the nsP1 coding sequence of SVMPA, which lead to amino acid changes, these revertants had retained the nt 127 and nt 963 mutations but had lost the nt 120 mutation. This, along with earlier findings, indicated that only the nt 127 and nt 963 mutations are required for resistance to MPA. This result also associated the nt 120 mutation with the host restriction phenotype. In other experiments derivatives of pToto1101 (a plasmid from which infectious Sindbis virus RNA can be transcribed) were constructed by site-directed mutagenesis and used to test the effect of specific mutations on the viral phenotype. Although we were unable to obtain viable virus with the nt 120 mutation alone, virus with the nt 120 and nt 127 mutations was viable and host-restricted. We suggest that the nt 120 mutation by itself is lethal and that the nt 127 mutation suppresses the lethal effect of the nt 120 mutation. The SVMPA mutation at nt 120, which is associated with the host range phenotype, changes Gln21 of nsP1 to Lys. When a more conservative change was engineered, i.e., to Asn, the virus was not host-restricted. Although the reason for the restriction of SVMPA replication in secondary CEF is not known, some possible explanations are discussed.

Unexpected isolation of virulent Newcastle disease virus from commercial embryonated fowls' eggs.

The authors report a case of causal isolation of virulent Newcastle disease virus (NDV) from embryonated eggs. The virus was isolated from uninfected chicken embryo liver and fibroblast cultures prepared from commercial embryonated fowls' eggs. A serological and virological investigation carried out on the breeders which had laid those eggs showed high titres against NDV, and virus isolation from cloacal swabs. The virus was also isolated from the progeny of the same breeders which showed no clinical signs of ND, following episodes of mortality. Vertical transmission of the virus is discussed.

Stimulation by insulin of protein synthesis in cultured chick embryo fibroblasts

Insulin stimulates protein synthesis in skeletal muscle of young animals and has been reported to exert similar effects on a variety of mammalian cell types in culture. However, with chick embryo fibroblasts we found that the extent of this effect was very sensitive to the culture conditions of the cells prior to the insulin treatment. The most reproducible results were obtained with cells that had been sub-cultured into medium in which fetal calf serum was replaced with 2% horse serum. Insulin only stimulated protein synthesis when added at supra-physiological concentrations. Insulin-like growth factor I was effective at much lower concentrations. Since chick embryo fibroblasts can be obtained in good yield, they offer, if treated under appropriate conditions, a suitable system for study of the mechanisms by which insulin and IGF-1 promote the initiation of protein synthesis in eukaryotic cells.

α5 Integrin Is a Critical Component of Adhesion Plaques in Myogenesis

We investigated the distribution and expression of α5β1 and α3β1 integrin in differentiating myogenic cells in culture. The myogenic cells expressed both α5 and α3 integrin subunits with the same molecular sizes as those expressed by chicken embryo fibroblasts (CEF). However, the ratio of total α5 to α3 was threefold higher in the muscle cultures than that in CEF cultures. A new method is described whereby adhesion plaque-associated integrin was cross-linked to its extracellular matrix ligand on the substrate using a nonpenetrating cross-linker, BS e, and integrin not involved in substrate adhesion as well as cytoskeletal proteins were removed with a zwitterionic detergent. α5 and β1 integrin, but not α3 could be cross-linked to fibronectin at adhesion plaques throughout myogenesis in culture. α5β1 integrin was found only at the edge of myoblasts 4 hr after plating but became distributed under their entire surface by 1 day in culture. When the muscle cells became elongated, a morphology they express after the initiation of terminal differentiation, and as they began to fuse, α5 was found redistributed in small adhesion plaques along the lateral edges of the postmitotic myocytes and early myotubes. In mature myotubes, which are large multinucleated branched structures, α5β1 integrin was localized to parallel streaks underneath their entire substrate surface. Throughout the different stages of myogenesis, vinculin colocalized with α5β1 integrin in adhesion plaques, but α-actinin only colocalized to the adhesion plaques in myoblasts, not in myotubes. These studies suggest that α5β1 integrin through its dynamic interaction with both fibronectin and the cytoskeleton is important for both the signals which initiate the differentiation process and for subsequent morphological and structural changes during the differentiation process.

Role of the 21-kDa protein TIMP-3 in oncogenic transformation of cultured chicken embryo fibroblasts.

The 21-kDa protein is an extracellular matrix (ECM) component whose synthesis is stimulated transiently during oncogenic transformation of chicken embryo fibroblasts (CEF) or after treatment of normal cells with the tumor promoter phorbol 12-myristate 13-acetate. Biochemical characterization indicates that the protein is related, but not identical, to two members of the family of tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2. The cDNA of the 21-kDa protein was recently cloned, and based upon its deduced amino acid sequence and other supporting data we propose that it is another member of this family, a TIMP-3. We now report electrophoretic purification of sufficient quantities of this protein to determine its function. The protein promotes the detachment of transforming cells from the ECM. Although its presence in the matrix may be necessary for cell release it is not the only factor involved because it does not influence the adhesive properties of nontransformed cells. It also appears to accelerate the morphological changes associated with cell transformation and stimulates the proliferation of growth-retarded, nontransformed cells maintained under low serum conditions. Based on these data we hypothesize that the 21-kDa protein promotes the development of the transformed phenotype in cultured cells.

In vitro evaluation of plasticizer activity on the growth and metabolism of chick embryo fibroblasts and on the development of chick embryo lungs.

Administration of di(2-ethylhexyl) phthalate (DEHP) to primary cultures of chick embryo fibroblasts brought about a decrease in cell proliferation rate after 48 h and an inhibition of both DNA and protein synthesis measured by [3H]thymidine and [3H]leucine, respectively, after 48 h. The growth of chick embryo lung rudiments in vitro was also depressed by DEHP treatment. Lung rudiment were smaller in DEHP-treated embryos after 6 days' treatment. These results indicate that DEHP has a cytostatic effect on embryonic cells and tissues.

Isolation and characterization of a novel type of growth factor derived from serum-free conditioned medium of chicken embryo fibroblasts.

A strong mitogenic activity for fibroblastic cells has been found in serum-free medium of growth-arrested primary cultures of chicken embryo fibroblasts (CEF). This serum-free conditioned medium promoted growth of NIH/3T3 cells and primary as well as secondary cultures of CEF. The mitogenic activity was as potent as 5% serum. Half-maximum stimulation was obtained with 20% of the initial concentration of the conditioned medium. The activity eluted at high M(r) (1-2 x 10(5)) from a gel-filtration column under nondenaturing conditions and was trypsin insensitive and thiol insensitive. Treatment with acid or urea converted the mitogen to a low-molecular-mass form, which showed a delayed induction of DNA synthesis. Purification of this factor (10000-fold) to apparent homogeneity was achieved by preparative isoelectric focusing, gel filtration, reverse-phase HPLC and nonreducing SDS/PAGE. The factor, termed CEF-derived growth factor (CDGF) was a 32-kDa, disulfide-linked heterodimer of a 15-kDa and a 17-kDa subunit as judged by SDS/PAGE, with a pI of approximately 7 in 8 M urea. It exhibited partial stability towards heat treatment and was trypsin sensitive. CDGF was active only in its dimeric form and half-maximum stimulation of NIH/3T3 cells was obtained at approximately 10 pM. The mitogenic activity was not suppressible by an antibody neutralizing the activity of transforming growth factor beta 1, 2 and 3 (TGF-beta). The physico-chemical properties suggest that CDGF is not identical with one of the common growth factors like fibroblast growth factor, platelet-derived growth factor, epidermal growth factor, insulin-like growth factor, or TGF-beta but rather represents a novel type of growth factor.

Isolation and Propagation of Infectious Bursal Disease Virus Using the Ovine Kidney Continuous Cell Line

Twenty-six samples known to contain infectious bursal disease virus (IBDV) were examined by virus-isolation attempts on ovine kidney (OK) cell line, Vero cell line, and chicken embryo fibroblast (CEF) cultures. Virus was isolated from two of 26 samples, three of 26 samples, and three of 25 samples on OK, Vero, and CEF cultures, respectively. However, in contrast to IBDV replication in Vero and CEF cultures, isolated virus was unable to induce serially sustained cytopathic effects (CPE) during successive passages in the OK cell line, unless cell lysates were treated with chloroform between every other passage. The cytopathogenicity of the untreated virus passaged in OK cells was revived and maintained upon passage in Vero cells. An initial single passage of laboratory or field material in OK cells followed by further passages in Vero cells resulted in virus isolation from six of 26 samples, which was better virus recovery than when either cell line was used alone or when CEF cultures were used. Twenty of the 26 test samples were originally positive when examined by nucleic acid hybridization with radiolabeled IBDV cDNA, indicating that some of the samples that were negative upon virus isolation using OK and Vero cells may have contained inactivated virus.

Effects of spermidine synthase inhibition in cultured chick embryo fibroblasts

The administration of bis-cyclohexylammonium sulfate (BCHS), a spermidine synthase inhibitor, to in vitro cultures of chick embryo fibroblasts caused a decrease in cellular spermidine levels and an increase in putrescine and spermine. Cell proliferation rate and DNA synthesis were also inhibited. As protein synthesis did not change, it would seem that low levels of cellular spermidine inhibit cell growth depressing DNA synthesis.

Safety of measles immunization in egg‐allergic children

Measles vaccines are prepared in chick embryo fibroblast culture and used throughout the world. Since 1963 dramatic reductions in the incidence of measles have been observed where the vaccination was extensively applied in the pediatric population. The best results were observed when the target population in the initial phase was that which produced over 90% of cases in the pre‐vaccinal era. A possible limiting factor to the diffusion of measles vaccination is the assumption that allergy to egg proteins is a contraindication. In this paper current knowledge about the safety of measles immunization in children with egg allergy is discussed.

Tenascin variants: differential binding to fibronectin and distinct distribution in cell cultures and tissues.

In the chicken, three tenascin variants have been characterized that are generated by alternative splicing of 3 of its 11 fibronectin type III repeats. Using monoclonal antibodies that react with common regions versus extra repeats of tenascin, we could distinguish and separate tenascin variants and investigate their interaction with fibronectin using multiple experimental procedures. Interestingly, in all assays used the smallest tenascin variant bound more strongly to fibronectin than the larger ones. These biochemical data were paralleled by the observation that in chick embryo fibroblast cultures only the smallest form of tenascin could be detected in the fibronectin-rich extracellular matrix network laid down by the cells. Furthermore, each tissue present in adult chicken gizzard contained a distinct set of tenascin variants. Those tissues particularly rich in extracellular matrix, such as the tendon, contained the smallest tenascin only. Intermediate-sized tenascin was present in smooth muscle, whereas the largest form was exclusively detectable underneath the epithelial lining of the villi. Thus it appears that cell type-specific forms of tenascin exist that are appropriate for the functional requirements of the respective extracellular matrices.