Embryo Kidney Cells Research Articles

Association of Reoviridae Particles in an Enteric Syndrome of Poults Observed in Turkey Flocks during 1988

An enteric syndrome of turkey poults, characterized by enteritis, crop mycosis, intestinal changes (pale, thin-walled ballooning with watery contents), and rickets, occurred during 1988 in 74 turkey flocks from different farms belonging to 9 California turkey growers. The flocks ranged in size from 9,000 to 120,000 birds. Pools of intestine sections from 618 birds, representing 78 field cases, were examined. Histopathological examination of the intestines showed a mild to severe atrophy with a reduced depth of crypts, which was more prominent in the distal part of the small intestine. Viral isolation attempts with primary cell cultures of chicken embryo kidney cells were negative. Examination by electron microscopy of negatively stained intestinal specimens revealed the presence of Reoviridae particles of 58.8 to 80 nm in diameter. Enzyme-linked immunosorbent assay results on the intestinal pools for mammalian and group A avian rotaviruses were negative. A statistically significant relationship was found for the presence of Reoviridae particles in the intestines of 10-21-day-old birds. Of the 7 most common pathological conditions analyzed, 2, rickets and intestinal changes (thin-walled ballooning intestine with watery contents), showed a statistically significant association with the presence of Reoviridae particles.

Cingulin: characterization and localization.

Cingulin, a protein component associated with the tight junctions of chicken intestinal epithelium, has been purified to homogeneity by a new procedure and characterized. Purified cingulin is a heat-stable elongated dimer, composed of two polypeptides of Mr 108,000 (cingulin-108), with a Stokes' radius of approximately 15 nm, and a molecular length of 130 nm +/- 32 nm. Monoclonal antibodies were used to determine the tissue distribution and subcellular localization of cingulin in a variety of avian tissues and cultured cells. Indirect immunofluorescence analysis of semi-thin frozen sections demonstrated that cingulin is localized in the junctional complex of various polarized epithelia and in the endothelium, whereas it is essentially absent from mesenchymal and myogenic cells. In permeabilized and fixed cultured chick embryo kidney cells, the antibodies stained solely the regions of contacts between the epithelial cells. Double immunofluorescent labeling of these cells with anti-cingulin and anti-vinculin antibodies showed that cingulin is localized close to the vinculin-rich cytoskeletal belt associated with adherens junctions, but is absent from focal contacts and stress fibers. In cultured kidney cells, actin was detected mainly in stress fibers and in the peripheral junctional regions, where it showed a distribution similar to that of cingulin, suggesting that actin filaments may be part of the submembrane cytoskeleton at the level of the tight junction. Indirect immunoelectron microscopic labeling of ultrathin frozen sections of chicken intestine showed that cingulin is localized along the endofacial surfaces of the tight junction (zonula occludens), and is apparently excluded from the more basal zonula adhaerens, and from the desmosomes.

Adenovirus vector expressing functional herpes simplex virus ICP0.

ICP0, one of the five immediate-early (IE) gene products of herpes simplex virus (HSV), is a potent activator of transcription. To assess the biological activities of ICP0 and to explore its mechanisms of action, two helper-independent recombinant adenoviruses were constructed. In each recombinant, the E1 region was substituted with the ICP0-encoding genomic segment under the control of either the adenovirus major late promoter (MLP-0) or the HSV IE-0 promoter (0PRO-0). Infection of HeLa cells or 293 cells (a human embryo kidney cell line expressing adenovirus 5 E1a and -b functions) with the MLP-0 recombinant results in the synthesis of more IE-0 mRNA and ICP0 protein than did infection with the 0PRO-0 recombinant. Although 293 cells infected with MLP-0 accumulate 5- to 10-fold more IE-0 mRNA late in the infection than cells infected with HSV, the level of the protein product, ICP0, increased only slightly. In 293 cells, both recombinants could replicate, albeit at a slower rate and with lower final yields than wild-type adenovirus. Neither virus replicates its DNA in HeLa cells, and thus ICP0 cannot substitute for adenovirus E1a; however, the level of ICP0 that accumulates in MLP-0-infected HeLa cells was comparable to that of HSV-infected HeLa cells. In a functional test, we demonstrated that the adeno-ICP0 recombinant viruses can transactivate a transfected TK-CAT cassette, indicating that the ICP0 is biologically active.

Serologic Comparison of Avian Reovirus Isolates Using Virus Neutralization and an Enzyme-Linked Immunosorbent Assay

The serologic relatedness of six avian reovirus isolates (CO8, S1133, 81-5, 2408, 1733, and UMI 203) were determined using a virus-neutralization (VN) test and an enzyme-linked immunosorbent assay (ELISA). Six groups of 20 specific-pathogen-free broilers each were twice infected with one of the six isolates per group. Serum was reacted against each isolate in a beta VN test in chicken embryo kidney cells and against the S1133 virus in ELISA. Relatedness (R) values, determined by cross VN, revealed that all belonged to a single serotype. However, the CO8 isolate represented a major subtype difference compared with the other isolates. R values among the five other isolates indicated minor or no subtype differences. ELISA also showed major differences between the CO8 and the other isolates.

Activation of the human beta interferon gene by the adenovirus type 12 E1B gene.

The transcription of endogenous beta interferon mRNA was activated in human embryo kidney (HEK) cells infected with adenovirus type 12 (Ad12) but was activated only inefficiently or not at all in HEK cells infected with Ad5 and rc-1 (Ad5 dl312 containing the Ad12 E1A region). The analysis with Ad12 mutants showed that Ad12 E1B products, especially the 19K protein, were important for the expression of the endogenous beta interferon gene and Ad12 E1A products were not involved in the expression. The expression of exogenously transfected pIFN-CAT (a hybrid plasmid having the human beta interferon promoter fused with the CAT gene) was activated in HEK and chicken embryo fibroblast (CEF) cells infected with either Ad12 or Ad5. The analysis of cotransfection of CEF cells with pIFN-CAT and plasmids containing fragments of Ad12 or Ad5 DNA showed that Ad12 or Ad5 E1B (possibly the 19K protein) was and E1A was not involved in the expression of the exogenous pIFN-CAT.

Quantitative ultrastructural study of nucleolus‐organizing regions at some stages of the cell cycle (G0 period, G2 period, mitosis)

The quantitative characteristics of chromosomal nucleolus-organizing regions (NORs) and some other nucleolar components were studied on ultra-thin sections of pig embryo kidney cells (PK cells). It was shown that: 1) nucleoli-per-cell volumes were 3 times smaller in the G0 period than in the G2 period; 2) the number of fibrillar centers (FCs) per cell in the G0 period, the G2 period, and at metaphase was equal to 7, 33.7, and 8, respectively; 3) mean volumes of individual FCs in the G0 period (0.033 +/- 0.005 micron3), G2 period (0.014 +/- 0.001 micron3), and at metaphase (0.025 +/- 0.002 micron3) were significantly different; 4) the total volumes of FCs calculated per haploid set of chromosomes were practically the same in the G0 (0.105 micron3) and G2 (0.107 micron3) periods, but were twice as large as those at metaphase (0.04-0.05 micron3). These data show that partial activation and inactivation of ribosomal genes in interphase PK cells are not accompanied by a considerable change in the total volume of FCs and may be due to the fragmentation and fusion of individual FCs. Complete inactivation of ribosomal genes in mitosis results in a decrease of total volumes of FCs per cell; 5) in G0 and G2 periods the total volume of the dense fibrillar component per nucleolus is practically proportional to the nucleolus volume (r = 0.99); 6) in the G2 period, the nucleolus volume is also proportional to the number of FCs (r = 0.99; 7) the volume of the dense fibrillar component within individual fibrillar complexes is not a constant one.(ABSTRACT TRUNCATED AT 250 WORDS)

Duck viral hepatitis type 1 vaccination: Monitoring of the immune response with a microneutralisation test in pekin duck embryo kidney cell cultures

A microneutralisation test in Pekin duck embryo kidney cell cultures with attenuated duck hepatitis virus (DHV) type 1 is described for monitoring and evaluating the immune response following vaccination of Pekin ducks against duck hepatitis type 1. Serum samples from 19 flocks of commercial fattening ducks 7 to 8 weeks of age had no detectable antibodies. Breeding ducks experienced exposure to DHV type 1 during the growing period as evidenced by some positive reactions in the VN test. Two consecutive subcutaneous vaccinations prior to the first breeding season resulted in high, and in most cases, uniform titres against DHV with small standard deviations and mostly narrow 95% confidence limits of the respective mean values. Additional vaccinations prior to further breeding seasons did not result in augmentation of the humoral immune response.

A Paramyxovirus of Serotype 3 Isolated from African and Australian Finches

Two virus isolates were obtained from exotic finches (Ortygospiza atricollis and Poephila cincta) suffering from apathy, diarrhea, conjunctivitis, and dysphagia. The isolates were identified as paramyxoviruses based on their multiplication characteristics in embryonating chicken eggs, chicken embryo fibroblasts, and chicken embryo kidney cell cultures, on morphology upon electron microscopy, and on other biological properties. Both isolates were serologically related to the reference strain of the paramyxovirus serotype 3. Intravenous infection of 42-day-old chicks resulted in no clinical signs, but intracerebral infection of 1-day-old chicks resulted in mortality and intracerebral pathogenicity indices of 0.25 to 0.35. Of five finches from various species inoculated with isolate 840/85, three remained clinically healthy through 6 weeks, but two died: one (Poephila cincta) 5 days postinoculation after showing nervous distress, and the other (Amandava amandava) suddenly 42 days postinoculation.

Induction of the stress response: alterations in membrane-associated transport systems and protein modification in heat shocked or Sindbis virus-infected cells

Heat shock or Sindbis virus infection of chick embryo (CE) or baby hamster kidney (BHK) cells resulted in a decrease in the uptake of 86Rubidium +, a K + tracer. Both stressful treatments decreased 86Rb + uptake by inhibition of the ouabain-sensitive Na +/K + ATPase. Alterations in the intracellular levels of mono-valent ions may be involved in translational or transcriptional control of the stress response. Heat shock or Sindbis virus infection also resulted in an increase in rate of uptake of [ 3H]deoxy- d-glucose and a decrease in the incorporation of [ 3H]glucosamine or [ 3H]mannose into most cellular proteins. These results suggested that heat shock or Sindbis virus infection alter hexose metabolism and that abnormally glycosylated proteins may accumulate in stressed cells. Exposure of uninfected chick embryo cells to elevated temperature had little effect on the overall rate of incorporation of [ 32P]orthophosphate into cellular proteins. However, one protein ( M r 31000; pp31) displayed increased incorporation of [ 32P]orthophosphate and two other proteins ( M r 33000 and 20000; pp33 and pp20) displayed decreased incorporation. Sindbis virus infection failed to mimic or to modify these heat shock induced alterations in protein phosphorylation.

Decrease in catalase activity of cultured cells by mycoplasma gallisepticum infection

The effect of Mycoplasma gallisepticum infection on the host cell catalase activity was histochemically examined in cultured chicken embryo fibroblasts (CEF) and kidney cells. The activity in normal cells was detected as fine, brown granules in the cytoplasm, which appeared ultrastructurally to correspond to anucleoid microbodies. By infecting cultured cells with a CEF-passaged strain of M. gallisepticum, the catalase-positive granules clearly decreased in amount, whereas the UV light-killed mycoplasma and the original strain failed to decrease the granules. The cell-passaged strain was able to induce cytopathic effects and this appeared to be due to its enhanced adherent ability as compared with the original strain. These findings suggest that attachment of viable organisms to cells is crucial to decrease the catalase activity and that the decreased activity may be an important process for the subsequent development of cytopathic effects.

Molecular cloning of the Mason-Pfizer monkey virus genome: Biological characterization of genome length clones and molecular comparisons to other retroviruses

The molecular cloning of the DNA provirus of Mason-Pfizer monkey virus (M-PMV) is described. Fourteen independent clones of integrated M-PMV proviruses were isolated from a human embryo kidney cell line that had been previously derived from a single cell clone infected with M-PMV. Characterization of these clones for size of insert, restriction pattern of flanking DNA, and presence of repetitive DNA in the flanking sequences revealed that 10 of the isolates were identical while the four remaining clones were unique. Three independent clones of unintegrated M-PMV proviruses containing a single copy of the long terminal repeat (LTR) were cloned from acutely infected human embryo kidney cells. Transfection assays revealed that 13 of 14 integrated proviruses and 2 of 3 unintegrated proviruses were capable of producing infectious virus. One of the integrated provirus clones (clone 6A) produced consistently higher titers of virus than all of the other clones in all assays used and in two different cell lines, indicating that it contained a mutation that enhances virus replication. The virus recovered after transfection was shown to be capable of inducing cell fusion in nontransformed cell lines, confirming that this property is associated with M-PMV. One of the clones was hybridized under conditions of varying stringency, to molecular clones of type B, C, and D retroviruses. These studies revealed M-PMV to be most closely related to squirrel monkey retrovirus (D-type virus) and more distantly related to mouse mammary tumor virus (B-type virus). Hybridization was also detected with clones from the pol gene region of a family of human endogenous sequences. No homology was detected with Rous sarcoma virus or most mammalian C-type viruses tested. The exceptions were baboon endogenous virus and RD114 in which previously identified homology in the env gene was confirmed. These results suggest that the type D and type B viruses can be linked together in a group of viruses of similar ancestral origin analogous to that recently proposed for the human T-cell leukemia viruses and bovine leukemia virus.

Transformation of dog embryo kidney cells by human herpesviruses

The infection of dog embryo kidney (DEK) cells with herpes simplex virus type 2 (HSV-2) or human cytomegalovirus (HCMV) led to the development of transformed cell lines. Rapidly dividing DEK cells with unlimited division potential exhibited growth in 2% serum, contained nuclear virus antigens, and formed small (±0.2 mm) colonies in 0.3% agarose. Immortal cell lines showing the same transformation properties were also obtained after transfection with purified HSV-2 or HCMV DNA. These results confirm the transforming capacity of both herpesviruses as well as the usefulness of this different type of mammalian cells in transformation studies.

Distinct regions of the human cytomegalovirus genome are responsible for the immortalization and tumorigenicity of animal cells

Dog embryo kidney cells were efficiently transformed by human cytomegalovirus (HCMV) particles or intact viral DNA. Negative results were obtained after transfection of the canine cells with recombinant plasmids carrying the HCMV Hind III-E subgenomic fragment or with limit Bgl II and Hind III digests of the viral genome. Immortalized dog cells with typical transformation properties appeared, however, after transfection with EcoR I fragments of the HCMV DNA. Distinct regions of the viral genome are probably responsible for the immortalization and the tumorigenicity of mammalian cells.

Modulation of the level of expression of cellular genes in adenovirus 12-infected and transformed human cells.

The level of expression of thymidine kinase (TK), heat shock protein 70 (HSP70), beta-tubulin and p53 was assessed in human embryo kidney cells (HEKs) infected with adenovirus type 12 (Ad 12) and Ad 12 early region 1 (E1) mutants. HSP70, beta-tubulin and p53 levels were unchanged but TK activity was dramatically increased following wild-type infection. The initial activation of TK required the expression of the product of the E1A 13S mRNA but sustained expression only occurred with those viruses expressing the E1B proteins as well. A number of human cell lines transformed with either Ad 12 or Ad 5 E1 DNA were also assessed for the level of expression of HSP70, beta-tubulin and p53. Both HSP70 and beta-tubulin levels were greatly increased compared with primary human cells although there was considerable variation between lines. p53 was only expressed at high levels in Ad 12-transformed lines expressing E1A and E1B proteins.

The adenovirus type 12 early-region 1B 58,000-Mr gene product is required for viral DNA synthesis and for initiation of cell transformation.

An E1B 58K mutant of adenovirus type 12 (Ad12), dl207, was constructed by the deletion of 852 base pairs in the E1B 58K coding region. The mutant could grow efficiently in 293E1 cells but not in HeLa, KB, or human embryo kidney (HEK) cells. Viral DNA replication of dl207 was not detected in HeLa and KB cells and was seldom detected in HEK cells. Analysis of viral DNA synthesis in vitro showed that the Ad12-DNA-protein complex replicated by using the nuclear extract from Ad12 wild-type (WT)-infected HeLa cells but not by using the nuclear extract from dl207-infected cells. In dl207-infected HeLa and KB cells, early mRNAs were detected, but late mRNAs were not detected. The mutant induced fewer transformed foci than the WT in rat 3Y1 cells. Cells transformed by dl207 could grow efficiently in fluid medium, form colonies in soft agar culture, and induce tumors in rats transplanted with the transformed cells at the same efficiency as WT-transformed cells. Tumors were induced in hamsters injected with WT virions but were not induced in hamsters injected with dl207 virions. The results indicate that the E1B 58K protein is required both for viral DNA replication in productive infection and for initiation of cell transformation, but not for maintenance of the transformed phenotype.

An assay for duck hepatitis virus type I in duck embryo liver cells and a comparison with other assays

Plaque assays for virulent and attenuated duck hepatitis virus (DHV) type I in duck embryo liver (DEL) and duck embryo kidney (DEK) cells are described. The results of assays for DHV type I strains by in vitro, in ovo and in vivo methods are compared. DHV type II and III did not produce plaques in DEL or DEK cells. A serological relationship between DHV type I and DHV type II and III could not be demonstrated by the plaque reduction neutralisation assay.

An electron microscopic study of nucleolus-organizing regions at some stages of the cell cycle (G0 period, G2 period, mitosis).

Nucleolus-organizing regions (NORs) at some stages of the cell cycle (G0 period, G2 period and mitosis), differing in the level of NOR transcription activity, were studied on ultra-thin serial sections of pig embryo kidney cells. Particular attention was given to the spatial organization of fibrillar centers (FCs) in the nucleoli and to comparing the number of FCs with that of NORs. It was shown that within nucleoli, FCs may be located singly or form groups surrounded by a dense fibrillar component. The overall number of FCs exceeds that of NORs 1.7-fold in the G0 period, and 4.6-fold in the G2 period. At prophase, with progressive condensation of chromosomes and NOR inactivation, the number of FCs decreases and at metaphase corresponds to the number of silver-stained NORs. The data thus obtained make it possible to conclude that in active nucleoli, the number of FCs is higher than that of NORs. Proceeding from these numerical and ultrastructural data, it is suggested that a single NOR of pig embryo kidney cells corresponds to one "fibrillar complex", i.e., to an association of a different number of FCs with the dense fibrillar component. These "fibrillar complexes" contain 1 to 3 FCs in the G0 period and 1 to 12 FCs in the G2 period.

Changes in intracellular pH induced by hyperthermia and hypoxia.

The effect of hyperthermia on pH of cells cultivated at varying pH of the medium and under different conditions of oxygenation has been studied. Chinese hamster fibroblasts and pig embryo kidney cells were used. The intracellular pH (pHi) was determined by the microfluorimetric method using fluorescein diacetate. The required temperature (37 degrees C and 41 degrees C) was maintained by a warm stage in combination with a thermochamber. Different conditions of oxygenation were achieved by placing the cells into an open (euoxic) or closed (hypoxic) chamber. Both hyperthermia and hypoxia are shown to result in the acidification of cells. The effect increases with the duration of exposure to the agents used. The decrease of pHi is more pronounced when hyperthermia is combined with hypoxia or when the pH of the medium is low. The results obtained correlate with the magnitude of directly damaging and radiosensitizing effects of hyperthermia being dependent on the environmental conditions.

Differential requirement for SV40 early genes in immortalization and transformation of primary rat and human embryonic cells

A series of recombinant plasmids carrying various DNA fragments of SV40 early region were used to test for their ability to immortalize primary cultures of rat embryo (RE) and human embryonic kidney (HEK) cells. When primary RE cells were transfected with plasmids containing an entire early region of wild-type SV40- or a deletion mutant in the small tumor (t) antigen, dl 1410-DNA, they were all immortalized. The immortalized cells could grow in soft-agar medium and produced large tumor (T)-antigen. Cultured RE cells transfected with pW2-t, which contains a deletion in the large-T-specific coding region, also gave rise to continuous cell lines. Interestingly, two of nine RE lines immortalized by pW2-t could also grow in soft-agar medium. The plasmid pW-t8 carrying a similar fragment of SV40 DNA as pW2-t, but lacking the processing and polyadenylation signal sequences, also immortalized RE cells. Surprisingly, the plasmid pD-t1 which contains neither the intact large-T nor the small-t function also immortalized RE cells. However, the RE lines immortalized by pW-t8 or pD-t1 were unable to grow in soft-agar medium and displayed a wide range of growth phenotypes. On the contrary, when primary HEK cells were used for immortalization experiments, only those SV40 plasmids carrying the intact large-T function were able to generate immortalized lines. The growth properties of these immortalized HEK lines can be categorized into two groups. Those HEK lines immortalized by the large T alone grew slightly denser and rounder than their parental normal HEK cells, while those immortalized by both the large-T and small-t antigens grew extremely fast, reached higher density, piled up on each other, and were anchorage independent. In addition, when these SV40 plasmids were used to directly transform primary HEK cells by the focus assay, the large-T clone, pD3-05, only transformed HEK cells to form light foci. Transfection by the large-T plus the small-t sequences either in cis or in trans, did increase the frequency of focus formation, and gave rise to dense foci which could grow in soft-agar medium.

Evaluation of chicken kidney and chicken embryo kidney cultures for the large-scale growth of attenuated influenza virus master strain A/Ann/Arbor/6/60- ca

Primary chicken kidney (CK) and chicken embryo kidney (CEK) cells were evaluated as possible substrates for growth of the cold-adapted attenuated influenza vaccine master strain A/Ann Arbor/6/60 (A/AA/6/60-ca). Yields of 10(6)-10(7) TCID50 per ml of culture fluid were obtained in either cell type. Yields from the human diploid strain MRC-5 were approximately 100-fold less. More reproducible cultures were obtained from CEK cells, using an overnight trypsinization step at 4 degrees C, than from CK cells. Comparable yields per embryo were obtained from CEK cells grown in roller cultures to those grown on the surface of microcarriers. These yields were less than those obtained from the allantoic fluids of whole embryos. Frozen storage of CEK or CK cells, after primary trypsinization, dispersal from a cultured CK primary monolayer or culture on microcarriers, was unsuccessful. The cold-adapted phenotype of A/AA/6/60-ca was retained after growth in CEK cultures and no differences in immunogenicity were detectable in mice between CEK- and allantoic-grown virus. Allantoic-grown preparations of A/AA/6/60-ca contained a lower protein concentration per infectious unit than those grown in CEK.