Rabbit Kidney Cell Line Research Articles

Isolation of equine herpesvirus type 5 in New Zealand

Aim. To report the first isolation of equine herpesvirus 5 (EHV-5) in New Zealand as part of a study of equine respiratory viruses in New Zealand. Methods. Nasal swabs and peripheral blood leukocytes were collected from 114 foals and adult horses, inoculated on to equine fetal kidney, rabbit kidney and Vero cell lines and observed for cytopathic effect. EHV-5 isolates were identified using an EHV-5 specific polymerase chain reaction. All samples positive for EHV-5 were also checked for the presence of EHV-2, EHV-1 or EHV-4 DNA using published type-specific primers. The polymerase chain reaction results were further confirmed by dot blot and Southern hybridisation with specific DIG-labelled probes. Results. EHV-5 was isolated from nasal swabs or peripheral blood leukocytes of 38 out of 114 horses sampled. From horses sampled more than once, EHV-5 was often isolated on more than one occasion. Most of the horses were infected with both EHV-2 and EHV-5 viruses. It was not possible to make an association between EHV-5 isolation and the presence of respiratory disease. Conclusion. EHV-5 is present in the New Zealand horse population. The exact role it plays in causing, or predisposing to, respiratory disease remains to be elucidated.

Tetracycline‐controlled expression of glycosylated porcine interferon‐gamma in mammalian cells

Tetracycline‐controlled expression plasmids that allow inducible expression of proteins in mammalian cells (Gossen & Bujard, 1992), have been used to express porcine interferon‐γ in the RK‐13 rabbit kidney cell line. Following neomycin selection, stable clones produced recombinant, glycosylated porcine interferon‐γ (rGPoIFN‐γ) only after removal of tetracycline (Tc). Southern blot analysis of one clone showed that approximately 50 copies of IFN‐γ cDNA were present in the cell genome. In the absence of Tc, stable clones secreted large amounts of rGPoIFN‐γ (up to 16 μg/ml) into the medium supplemented with 10% FCS and high glucose concentration. Molecular weight comparison of 35S‐Methionine, labelled rGPoIFN‐γ with natural leukocytic IFN‐γ after immunoprecipitation, revealed 4 major glycoforms with apparent Mr of 27,000; 25,000; 20,000 and 18,500, that are almost identical in both IFN‐γ species. In both cases, all 4 glycoforms resolved into 2 polypeptide monomers with apparent Mr of 16,500 and 14,500 upon deglycosylation with N‐glycosydase F. The biological activity of rGPoIFN‐γ was in the same range as that of natural leukocytic PoIFN‐γ (2 × 106 U/mg). Eventually, this recombinant mammalian IFN‐γ should constitute a very useful substitute for leukocyte PoIFN‐γ in in vitro or in vivo experiments.

Construction and characterization of an equine herpesvirus 1 glycoprotein C negative mutant

An equine herpesvirus 1 (EHV-1) strain RacL11 mutant was constructed that carries the Escherichia coli LacZ gene instead of the open reading frame encoding glycoprotein C (gC). The engineered virus mutant (L11ΔgC) lacked codons 46–440 of the 1404 bp gene. On rabbit kidney cell line Rk 13 and equine dermal cell line Edmin337, the L11ΔgC virus grew to titers which were reduced by approximately 5- to 10-fold compared with wild-type RacL11 virus or a repaired virus (R-L11ΔgC). However, when L11ΔgC growth properties were analyzed on primary equine cells a decrease of viral titers was observed such that extracellular L11ΔgC titers were reduced by 48- to 210-fold compared with those of wild-type or repaired virus. Heparin sensitive and heparin resistant attachment was assessed by binding studies using radiolabeled virion preparations. These studies revealed that EHV-1 gC is important for heparin sensitive attachment to the target cell. Similar results were obtained when cellular glycosaminoglycan (GAG) synthesis was inhibited by chlorate treatment or when cells defective in GAG synthesis were used. L11ΔgC also exhibited significantly delayed penetration kinetics on Rk 13 and primary equine cells. Infection of mice with L11ΔgC did not cause EHV-1-related disease, whereas mice infected with either RacL11 or R-L11ΔgC exhibited massive bodyweight losses, high virus titers in the lungs, and viremia. Taken together, EHV-1 gC was shown to play important roles in the early steps of infection and in release of virions, especially in primary equine cells, and contributes to EHV-1 virulence.

Complementation of a Replication-Defective Mutant of Equine Herpesvirus Type 1 by a Cell Line Expressing the Immediate-Early Protein

Equine herpesvirus type 1 (EHV-1) possesses a sole, diploid immediate-early (IE) gene that encodes a major regulatory protein of 1487 amino acids capable of modulating expression of both early and late EHV-1 promoters and capable oftrans-repressing its own promoter. In this study, a rabbit kidney cell line (IE13.1) that constitutively expresses the EHV-1 IE protein was generated by cotransfection of rabbit kidney (RK-13) cells with the viral IE gene and a neomycin resistance marker. The IE protein expressed by this cell line was shown (1) to be expressed by and to localize to the nucleus of virtually all cells as demonstrated by indirect immunofluorescence, (2) to be the full-size IE polypeptide as judged by Western immunoblot analyses with an anti-IE protein-specific antibody, and (3) to be functional as shown by the transactivation of two representative EHV-1 early promoters linked to the chloramphenicol acetyltransferase reporter gene in transient transfection assays. The IE13.1 cell line was able to complement a recombinant virus in which both copies of the IE gene were replaced by insertion of theEscherichia coli lacZgene. This IE deletion mutant, designated KyAΔIE, was not able to replicate in equine, rabbit, or mouse cells but was capable of replication in the IE13.1 cells that provided the IE protein intrans. Rescue of the KyAΔIE virus was achieved by recombination with a marker plasmid that harbors the wild-type IE gene, and the rescued virus (KyAΔIER) was able to grow on noncomplementary cells. Overall, these results offer direct evidence that the IE gene is essential for EHV-1 replication and provide reagents useful for the analysis of IE protein function.

Construction of a vaccinia virus deficient in the essential DNA repair enzyme uracil DNA glycosylase by a complementing cell line.

The vaccinia virus D4R open reading frame, encoding the essential DNA repair enzyme uracil DNA glycosylase, was expressed in two permanent cell lines, the rabbit kidney cell line RK13 and the human fibroblast cell line 293. The temperature-sensitive vaccinia virus mutant ts4149, which maps within D4R, was able to grow under restrictive conditions in both of these transformed cell lines. Cell clones complemented D4R function to various degrees, demonstrating complementation of an essential vaccinia virus gene by a cell line constitutively expressing the essential function. Thus, the complementing host cells allowed the rescue of a virus defective in the D4R gene, demonstrating that this system may be used for the propagation of defective cytoplasmic DNA viruses. The defective virus grew to high yields only in the engineered cell lines. The data support the hypothesis that early gene products, such as uracil DNA glycosylase, supplied in trans can fully complement essential viral functions.

23 Phenotypic characterization of the rabbit kidney cell line LLC-RK1. implication for pharmacotoxicological studies

23 Phenotypic characterization of the rabbit kidney cell line LLC-RK1. implication for pharmacotoxicological studies

Comparative toxicity of oral cephalosporin antibiotics in the rabbit and in a rabbit kidney cell line (LLC-RK 1)

The nephrotoxic potential of four oral cephalosporin antibiotics, cephalexin, cefaclor, LY195885 and LY171217, was determined in rabbits given single oral doses of 250–500 mg/kg body weight. Histopathological changes, blood chemistry, and ex vivo renal slice function were evaluated 48 hr after dosing. Additionally, the viability of rabbit renal cells in culture (LLC-RK 1) was evaluated by nigrosin dye exclusion after 48 hr exposure to each antibiotic at concentrations of 0.5–2.0 mg/ml. Only LY171217 was significantly nephrotoxic in vivo. Prominent lesions were observed at 500 mg/kg body weight and were accompanied by marked increases in blood urea nitrogen and serum creatinine, and decreases in ex vivo renal slice gluconeogenesis and p-aminohippurate and tetraethylammonium uptake. In vitro toxicity to renal cells correlated well with the in vivo results yielding TC 50 values (TC 50 = concentration producing 50% lethality) > 1.0 mg/ml for cephalexin, LY195885 and cefaclor. LY171217, on the other hand, was significantly toxic in vitro (TC 50 = < 0.5). These results suggest that renal cells in culture may provide a useful method for examining the nephrotoxic potential of oral cephalosporins before in vivo studies.

In vitro toxicity of antibiotics to LLC-RK 1 rabbit kidney cells

The renal toxicity of cephalosporin and aminoglycoside antibiotics has been studied in an in vitro system using the rabbit kidney cell line LLC-RK 1. The effect of a rabbit kidney S-9 metabolizing system on the toxicity the antibiotics was also studied. Cultures of the LLC-RK 1 cells were established in microtitre plates and exposed to test compounds in the concentration range 0 to 2000 μg/ml, either in the presence or absence of an S-9 metabolizing system. The compounds were in contact with the cultures for a period of 48 hr before viability of the cells was determined. Assay of cell viability was based on the mitochondrial conversion of 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide to a coloured formazan product (MTT Assay). Duplicate cultures were also tested with a neutral red staining assay. Cephalosporins tested were cephaloridine, cephalothin, cephapirin, cefotaxime, ceftazidime, cephaloglycin, cefoperazone and cefazolin. The aminoglycoside antibiotics tested were gentamicin, neomycin and kanamycin. A good correlation was seen between in vitro toxicity and in vivo effects for these compounds. Further techniques were investigated to assess sub-cytolethal impairment of renal function in the LLC-RK 1 cells. Both in vivo clearance studies and in vitro kidney slice methods have used transport of the organic ions tetraethyl ammonium and p-aminohippurate as indices of renal function. Therefore the ability of LLC-RK 1 cells to transport these ions was investigated. Where ion transport was demonstrated the effect of nephrotoxic antibiotics on this kidney function was studied.

Viral transcripts in cells infected with defective interfering particles of equine herpesvirus type 1

Equine herpesvirus type 1 (EHV-1) preparations enriched in defective interfering particles (DIPs) have previously been demonstrated to mediate the coestablishment of persistent infection and oncogenic transformation in primary hamster embryo fibroblasts. In this study, it was demonstrated that infection of a rabbit kidney (RK) cell line with EHV-1 DIP-enriched preparations also results in the establishment of persistent infection. Viral transcription was characterized in RK cells infected with DIP-enriched stocks and compared to viral transcription in RK cells infected with standard (STD) EHV-1. During the first 8 hr of infection with the DIP-enriched EHV-1 preparation, viral DNA sequences which are conserved in the DIP genome were predominantly expressed. Thus, these transcripts originate from DNA sequences that contain the components of the defective genome which originates from DNA sequences mapping at 0.00–0.04 of the Long region terminus and within two portions of the Short region inverted repeats (IR), 0.78–0.79 and 0.83–0.865 of the internal IRs and 0.99–1.00 and 0.915–0.95 of the terminal IRs. The overwhelming majority of viral transcripts that were synthesized in the DIP-enriched infections appeared to correspond to transcripts expressed in STD infection as assessed by Northern hybridization analysis but the synthesis of transcripts originating from sequences not conserved in the defective genome was significantly delayed. However, some high molecular weight RNA species that were synthesized in STD infections were not detected in DIP-enriched infections. Studies utilizing metabolic inhibitors indicated that viral transcription in DIP-enriched infections, like that of STD cytocidal infection, is regulated in an immediate early, early, and late manner.

Regulated expression of foreign genes in mammalian cells under the control of coliphage T3 RNA polymerase and lac repressor.

Systems that stringently regulate the expression of individual genes within a complex genetic background have contributed greatly to the analysis of gene function. In this report the development of a highly regulated expression system in mammalian cells is described in which transcription of a foreign gene is mediated by the bacteriophage T3 RNA polymerase under the control of the Escherichia coli lac repressor. Rabbit kidney cell lines have been established that constitutively express the phage RNA polymerase and lac repressor. The two bacterial proteins regulate the transcription of the coding sequence of the firefly luciferase, which has been placed under the control of a T3 promoter/lac operator fusion. In the presence of the inducer isopropyl beta-D-thiogalactoside, efficient T3 polymerase-dependent transcription is observed, which is tightly repressed in the absence of inducer. Translation of the T3 transcripts can be mediated by vaccinia virus functions. The demonstration that a specific transcription activity can be regulated over a range of several orders of magnitude in higher eukaryotic cells by using a highly specific and nontoxic inducer has broad implications for a variety of studies.

Establishment of an ELISA for the detection of orthopox viruses based on neutralizing monoclonal and polyclonal antibodies.

Various combinations of polyclonal and neutralizing monoclonal antibodies (MAbs) of distinct specificity were evaluated as capture or detecting antibodies in an orthopox virus antigen ELISA. Acceptable results were achieved in an assay based on polyclonal antibodies. A 10 times higher sensitivity, however, was obtained using a combination of one monoclonal catching antibody reactive with viral envelope epitopes and polyclonal detection antibodies. This configuration proved to be superior in sensitivity to all others. Specificity was confirmed with 5 vaccinia virus strains (including one recombinant virus) and 8 species of orthopox viruses. Monkeypox and mousepox virus reacted exclusively in the polyclonal assay due to a lack of the specific epitope for the monoclonal antibody. The detection limit compared to the infectivity titer amounted to 10(3)-10(4) in the monoclonal/polyclonal and to 10(4)-10(5) TCID50/0.1 ml in the polyclonal combination. The correlation between infectivity- and ELISA-titer was demonstrated by a study of the replication cycle of the rabbitpox virus Uetrecht in the permanent rabbit kidney cell line RK-13. With the established ELISAs vaccinia virus could also be detected in organ suspensions of lethally infected NMRI-mice, depending on the level of infectivity.

An adenovirus infection of the kidney of Franklin's ground squirrels (Spermophilus franklini) in Saskatchewan, Canada.

During routine pathological studies of Franklin's ground squirrels (Spermophilus franklini) collected during a predator control program, basophilic intranuclear inclusions were found in the collecting tubule epithelium of the renal papillae in seven of 13 squirrels. This was associated with marked karyomegaly in affected cells. An inflammatory response was not seen in the adjacent tissues. Electron microscopic examination of affected cells demonstrated that the enlarged nuclei contained numerous virus-like particles. Autoculture and serial passage of renal medullary cells resulted in the isolation of virus particles producing intranuclear inclusions and cytopathic effect. The virus possessed properties typical of adenoviruses, but showed no evidence of hemagglutinating activity with a range of species of erythrocytes tested under several temperature conditions. The isolates were relatively host-cell specific; they failed to grow in hamster and rabbit kidney cell lines and in ground squirrel kidney cortical cells.

The Mechanism of the Antiviral Effect of Interferon Differs in Two Strains of a Rabbit Kidney Cell Line

Two variant strains of a rabbit kidney cell line, RK-13, differ markedly in their interferon (IFN)-mediated antiviral mechanism. Both strains are permissive for the growth of vesicular stomatitis (VSV) and vaccinia viruses. Following treatment with 100 U/ml of rabbit IFN, both cell strains restrict VSV yield about a 1000-fold; vaccinia is not inhibited by IFN in either cell strain. However, vaccinia is able to rescue VSV from the inhibitory effects of IFN in one strain (RK-13), but not in the other strain of cells (RK-13-37). Investigation of the mechanism of this phenomenon reveals that VSV growth is blocked at different levels in the two cell strains. In the strain in which vaccinia can rescue VSV from the effects of IFN (RK-13), the major IFN-mediated restriction of VSV growth is at the level of protein synthesis; in the other strain in which vaccinia is unable to rescue VSV (RK-13-37), IFN restricts VSV at earlier steps, including penetration and primary transcription. The two cell strains provide useful models for studying the multifaceted nature of IFN-mediated antiviral effects.

Specificity and partial purification of a factor in spent media from sertoli cell-enriched cultures that stimulates steroidogenesis in leydig cells

There is increasing evidence that factors derived from the seminiferous tubules influence Leydig cell function in a paracrine way. In previous experiments we demonstrated that conditioned media from Sertoli cell-enriched cultures contain a protein with stimulatory activity on prepubertal rat Leydig cells. In this paper we further studied the specificity of this factor. In addition we describe a simple but efficient partial purification procedure. It is demonstrated that Sertoli cell conditioned media contain a factor that stimulates the testosterone ouput from prepubertal and adult Leydig cells. The effects are evident within the first hour of incubation and can be observed in the presence as well as in the absence of LH. Peritubular cells do not produce a similar factor but enhance the production of the Leydig cell stimulating factor when cocultured with Sertoli cells. The Sertoli cell factor acts on rat as well as on mouse Leydig cells. It barely influences the adrenostenedione output of ovarian stromal cells or the corticosterone output of adrenal cells. The production of this factor is enhanced by dbcAMP, FSH, l-isoproterenol and glucagon but is not affected by androgens. The characteristics of the Sertoli cell factor have been compared with those of a Leydig cell stimulating factor in the medium from an established rabbit kidney cell line: RK 13. It is shown that the active principle in RK 13 conditioned medium is also a thermolabile trypsin-sensitive protein with a mol. wt of more than 10,000. Nonetheless, the RK 13 and Sertoli cell derived factors act by different mechanisms since at maximally effective concentrations their effects are additive. Finally it is demonstrated that molecular weight fractionation of Sertoli cell conditioned medium using an Amicon ultrafiltration system results in a 50- to 130-fold increase in Sertoli cell factor activity in a fraction corresponding to a mol. wt of 10,000 up to 30,000.

Effects of peptides cleaved from human fibrinogen by plasmin on rabbit kidney cells in culture

Low molecular weight fibrinogen degradation products (LMW-FDP) containing a mixture of dialysable peptides cleaved from human fibrinogen by plasmin are cytotoxic to an established line of rabbit kidney cells and to primary cultures of rabbit kidney cells. The presence of LMW-FDP in a concentration of 50 μg/ml during the cell cultivation caused a considerable release of 51Cr from prelabelled cells and inhibited 3H-thymidine and 86Rb uptake. Among three isolated peptides of established primary structure only one, 6D: Ser-Gln-Leu-Gln-Lys-Val-Pro-Pro-Glu-Trp-Lys, induced a signifificant effect, i.e. it enhanced 3H-thymidine incorporation. Two others, 6A: Ala-Arg-Pro-Ala-Lys and 6E: Thr-Ser-Glu-Val-Lys, did not influence the examined parameters. Hence other components of LMW-FDP must be assumed to be responsible for the cytotoxic effect on kidney cell cultures.

Rubella Virus Plague Neutralization Test…Description of Assay and Comparison with Alternative Rubella Antibody Determination after Vaccination…

A reproducible and sensitive rubella plaque neutralization test was established utilizing a Japanese, live, attenuated vaccine strain (KRT-Kitasato, GMK 4/RT 36/RK 1) in a rabbit kidney cell line (RK-13). Rubella strain, cell line derivation, addition of complement and temperature of incubation were all critical to the efficiency of plaque formation. Two hundred sera from 25 children collected longitudinally following rubella vaccination with HPV77DE5 were tested for rubella antibody by three different assays: 1) plaque neutralization, 2) hemagglutination inhibition, and 3) enzyme linked immunoadsorbent assay by commercial kit. Plaque neutralization antibody appeared more slowly after immunization than that determined by hemagglutination inhibition, but persisted in all 22 successfully immunized children over a five to seven year period. The same 22 children had a rise in hemaggulutination inhibition antibody, but lost detectable antibody five to seven years after vaccination. Only 21 children seroconverted by enzyme immunoadsorbent assay. Nine of these lost detectable antibody by five years.

Establishment of rotavirus persistent infection in cell culture. Brief report.

Inoculation of the rabbit kidney cell line (RK13) with simian rotavirus SA11 resulted in persistently infected (carrier) cultures. A small percentage of these cells produced infectious virus (> 25 passages) and trypsin treatment enhanced virus production.

Metabolism of rabbit kidney cells incubated in vitro with phospholipase C from Staphylococcus aureus and Clostridium perfringens

An established cell line of rabbit kidney cells (RK-13) was treated with subcytotoxic concentrations of two highly purified toxic bacterial phospholipases C, beta-toxin from Staphylococcus aureus and alpha-toxin from Clostridium perfringens. Although the concentrations used did not increase the number of cells stained with nigrosin, a decrease in active uptake of 86Rb and increase of leakage out of the cell of 86Rb was noted within 60 min of incubation at 37°C with 40 hemolytic units of beta-toxin and 10 enzymatic units of alpha-toxin. Binding of ouabain to the cells was also impaired. Cl. perfringens alpha-toxin caused in general much greater damage to the cells than the staphylococcal beta-toxin, which is in accordance with the broader substrate specificity of the former enzyme. Cell metabolism was also studied up to 8 hr after incubation with the toxins for 1 hr. Staphylococcal beta-toxin temporarily inhibited the uptake of 3H-glycine, but not 3H-thymidine or 3H-uridine. Cl. perfringens alpha-toxin, on the other hand, affected first the uptake of 3H-thymidine and later the uptake of 3H-glycine. The intracellular content of cyclic AMP was doubled after 1 hr, but declined in a few hr below the control value. It is concluded that the cytoplasmic membrane was affected by these two enzymatic toxins in subcytotoxic doses, and that the different membrane functions investigated varied as to their dependence upon an intact phospholipid milieu.

Persistent infection of cultured mammalian cells by Japanese encephalitis virus.

Persistent infections were established by serial undiluted passage of flavivirus Japanese encephalitis virus in a line of rabbit kidney cells (MA-111). The persistently infected cells resembled uninfected cells in most respects. Low levels of infectious virions were released from a small percentage of cells, and a larger and more variable percentage was shown to possess viral antigen by fluorescent-antibody staining. Released viruses were shown to interfere with replication of wild-type Japanese encephalitis virus. Persistently infected MA-111 cells could not be superinfected with homologous wild-type Japanese encephalitis virus but could be superinfected with two heterologous viruses. Transfer of cell culture medium from persistently infected MA-111 cells to a line of African green monkey kidney cells (Vero) resulted in similar persistent infections in the latter cells. Temperature sensitivity and host-cell interferon production were not involved in establishment or maintenance of persistence. Determination of ratios of physical particles to infectious particles revealed that many defective, noninfectious viruses were present, suggesting that defective interfering particles may be responsible for persistency.

Preliminary characterization of isoimmunogenic placental antigens in the rabbit.

By isoimmunization of adult virgin female rabbits with rabbit placental extracts we have demonstrated the production of humoral antibody against two antigens, Ag-1 and Ag-2. Neither antigen is a normal serum protein, nor are they allotype proteins. Ag-1 has an alpha-2 mobility and a molecular weights of about 400,000. Ag-2 exhibits a gamma mobility but was not characterized. The antibody activity directed to Ag-1 in immune sera was the complement-fixing IgG fraction. Direct fluorescence-labeled antibody technique revealed that Ag-1 is a ubiquitous intracellular antigen primarily localized in the nucleus. The fast-growing, SV-40 virus-transformed rabbit kidney cell line (TRK-1) expressed much higher levels of Ag-1 than the slow-growing, non-transformed normal rabbit kidney cell line (LLC-RK1). The possibility that elevation of Ag-1 production is associated with rapid cell proliferation is discussed.