Isolation of influenza virus in human lung embryonated fibroblast cells (MRC-5) from clinical samples.

Ninety-four pharyngeal swab samples corresponding to 94 patients with suspected influenza virus infection were inoculated in Madin-Darby canine kidney (MDCK) cells, the conventional cell system for the isolation of influenza virus, and in fibroblastic human embryo lung (MRC-5) cells, a cell system less commonly used for this purpose but one frequently used in clinical virology laboratories. Both cell preparations were treated with trypsin. Influenza virus was recovered from 15% of the samples inoculated in MDCK cells and from 18% of those inoculated in MRC-5 cells. The use of MRC-5 cells can simplify the search for respiratory viruses and would assist in the rapid detection of influenza virus during new epidemics.     

Altered glycosylation in Madin-Darby canine kidney (MDCK) cells after transformation by murine sarcoma virus

The changes in glycosylation of an immortalized epithelial cell line (MDCK) before and after progression towards a more malignant phenotype have been studied. The parental MDCK-3 cells were immortalized after long-term passage in vitro and have shown no tendency for spontaneous acquisition of malignancy-related phenotypes such as tumorigenicity, invasion and metastasis. They conserved morphological and functional characteristics of the epithelial tissue of origin. The ras-MDCK cells acquired the fully malignant phenotype after transformation with a Harvey murine sarcoma virus; they were immortalized, invasive in vitro and produced invasive and also metastatic tumors after subcutaneous injection into nude mice. Using immobilized lectins and gel chromatography, before and after liberation of O-linked glycans from the peptide moieties and also after removal of terminal sialic acid, we have found differences in the glycosylpeptides of both whole cells and cell surface trypsinates from ras-MDCK cultures as compared to the parental MDCK-3 cultures: (i) more sialic acid in the N-linked tri- and tetra-antennary structures; (ii) more fucosylation in the N-glycosylpeptides; (iii) more bi-antennary N-glycosylpeptides and less O-linked glycans; and (iv) a lower molecular weight of the O-linked glycans probably due to a decreased sialylation. It is concluded that alterations in sialylation and fucosylation of the cell surface exposed glycans accompanied progression of MDCK-3 cells towards a more malignant phenotype.     

The multiplication of an influenza C virus in an established line of canine kidney (MDCK) cells

Mink lung cells (MvILu) are highly susceptible to varicella-zoster virus (VZV). The titres of cell-free VZV suspensions reached 1.0 x 10(7) p.f.u./ml at 3 days post-infection, with subsequent cell degeneration, if MvILu cells were infected with a multiplicity of infectious virus of 0.01 p.f.u./cell. In contrast, during the same period and under the same conditions the titres of cell-free VZV were 10(2) to 10(3) times lower when grown on human foreskin fibroblasts. A fast and reliable plaque assay and a neutralization test for VZV on MvILU cells, were developed.     

Permanent canine kidney (MDCK) cells for isolation and plaque assay of influenza B viruses

A wide range of influenza B virus strains with various passage histories uniformly formed well-defined clear plaques with high efficiency in cultures of an established line of canine kidney cells (MDCK). PFU titers of the viruses assayed in MDCK exceeded the titers assayedin ovo. With recently isolated strains such as B/Hong Kong/5/72 and Gifu/2/73, the PFU/EID₅₀ ratios were as high as 100 to 400. MDCK cells have been successfully employed for primary isolation of influenza B viruses from throat washings of patients by direct plaquing.     

A simplified plaque assay for influenza viruses in Madin-Darby kidney (MDCK) cells

A variety of influenza A and B viruses plaque in MDCK cell in trypsin is added only at the time of viral adsorption to the monolayer. Therefore, a conventional soft-agar overlay can be employed without addition of proteolytic enzymes. Plaquing efficiency was comparable to that when embryonated eggs were used to determine infectivity. Finally the method is simple and economical.     

Temperature-responsive culture dishes allow nonenzymatic harvest of differentiated Madin-Darby canine kidney (MDCK) cell sheets

We have developed a temperature-responsive culture dish grafted with a poly(N-isopropylacrylamide) (PIPAAm). Various types of cells adhere, spread, and proliferate on the grafted dishes in the presence of serum at 37 degrees C. By reducing only temperature, these cells can be harvested noninvasively from the dishes according to rapid hydration of the grafted polymer. Because the harvest does not need enzymatic digestion, differentiated cell phenotypes are retained. In the present study, a renal epithelial cell line, Madin-Darby canine kidney (MDCK) cell, was cultured on the dishes, and cell behavior was examined. MDCK cells showed differentiated phenotypes such as dome formation during long-term culture, similar to on ungrafted dishes. After 1-week culture at 37 degrees C, trypsin digestion disrupted cell-cell junctions but failed to liberate cells from both ungrafted and grafted dishes. However, short-term incubation at 20 degrees C released confluent MDCK cells as a single contiguous cell sheet only from the polymer-grafted dishes because of selective disruption of the cell-surface binding. Immunocytochemistry with anti-beta-catenin antibody revealed that functional cell-cell junctions were organized even in the recovered cell sheets. Intriguingly, incubation time at 20 degrees C required for cell sheet detachment gradually shortened during long-term culture before reducing temperature. The acceleration of cell detachment was correlated to the decrease of a single cell area by means of cell contractile force. These findings suggest that cell sheet detachment from PIPAAm-grafted dishes should be accomplished by both PIPAAm hydration and cellular metabolic activity such as cell contraction.     

Comparison of primary rabbit kidney and MRC-5 cells and two stain procedures for herpes simplex virus detection by a shell vial centrifugation method.

By using a conventional tissue culture method as a standard, four shell vial centrifugation culture (SVC) formats were compared for herpes simplex virus (HSV) detection in 300 clinical samples. Both MRC-5 and primary rabbit kidney (PRK) cells were used in the conventional and SVC systems. In addition, both a direct monoclonal fluorescent antibody to HSV (MAb-FA; Syva Corporation, Palo Alto, Calif.) and an indirect HSV polyclonal antibody-horseradish peroxidase stain (poly-HRP; Difco Laboratories, Detroit, Mich.) were used to stain shell vials of both cell types. Conventional tubes were incubated for up to 7 days with daily examination for cytopathic effect, which was confirmed as HSV by staining with an MAb-FA. Shell vials were inoculated, centrifuged, incubated for 16 to 24 h, and stained directly with MAb-FA or indirectly with a poly-HRP stain. Of the 300 specimens examined, 82 (27%) were HSV positive by conventional tissue culture. PRK cells detected 81 (99%) positive specimens, compared with 74 (90%) specimens detected with MRC-5 cells. Of the 82 positive specimens by conventional culture, the SVC formats detected 68 by MRC-5 and MAb-FA, 74 by MRC-5 and poly-HRP, 64 by PRK and MAb-FA, and 77 by PRK and poly-HRP. Therefore, PRK stained by an indirect method with poly-HRP was the most sensitive of the SVC formats tested, detecting 94% of the positive specimens.     

Mechanisms of dopamine effects on Na-K-ATPase activity in Madin-Darby canine kidney (MDCK) epithelial cells

Dopamine decreases tubular sodium reabsorption, attributed in part to Na-K-ATPase inhibition in the proximal convoluted tubule (PCT). Because the final regulation of sodium excretion occurs in the collecting duct, where specific dopamine DA1 binding sites have been demonstrated, we examined the effects of dopamine, as well as of DA1 and DA2 receptor agonists on Na-K-ATPase activity and on the number of units in Madin-Darby canine kidney (MDCK) cells, which retain differentiated properties of the renal cortical collecting tubule epithelium. Dopamine (10^–5 M) inhibited pump activity (by 50%) and reduced the number of units. This effect was reproduced by the DA1 agonist SKF 38393, which inhibited pump activity in a dose- and time-dependent manner (maximum, 10^–5 M). The DA2 agonist quinpirole hydrochloride was without effect, either alone or in combination with SKF 38393. Inhibition of pump activity by dopamine was totally abolished by H7 (100 M), an inhibitor of protein kinase (PK), but partially by 2, 5-dideoxyadenosine (DDA) and H4, respective inhibitors of cAMP production and PKA, which suggests that the dopamine effect on Na-K-ATPase activity may be linked to activation of both PKC and PKA. In these cells, amiloride addition during preincubation did not alter the effect of dopamine on Na-K-ATPase activity; in contrast, furosemide increased further the inhibitory effect of dopamine on the enzyme activity. Monensin addition (10^–3 M) reversed the inhibitory effect of dopamine after a 30-min preincubation. These results indicate that dopamine inhibits Na-K-ATPase activity in MDCK cells and that this inhibition is mediated by activation of the DA1 receptor, they also suggest that PKC and PKA activation inhibits apical sodium entry.     

Plaque assay and primary isolation of influenza a viruses in an established line of canine kidney cells (MDCK) in the presence of trypsin

A wide variety of influenza A viruses, comprising human, equine, porcine, and avian strains, grew productively in an established line of canine kidney cells (MDCK) under an overlay medium containing trypsin, and formed well-defined plaques regardless of their prior passage history. Plaquing efficiency was comparable to the efficiency of infection in fertile eggs via allantoic route. MDCK cells have also been successfully employed for the primary isolation of influenza A virus from throat washings of patients. Parallel titration of several clinical specimens showed that the inoculation into MDCK cells followed by incubation in the presence of trypsin was an isolation procedure as sensitive as the amniotic inoculation into fertile eggs.     

Diagnosis of respiratory tract viruses in 24 h by immunofluorescent staining of shell vial cultures containing Madin-Darby Canine Kidney (MDCK) cells.

Nine hundred and seventy-eight clinical specimens were examined taken from patients with respiratory tract viruses (RV)-like syndrome between November 1996 and July 1998. The study was undertaken to evaluate the effectiveness of centrifuge-enhanced shell vial cultures (SVC) containing Madin-Darby Canine Kidney (MDCK) cells, combined with immunofluorescent (IF) staining in 24 h. This technique rapidly detects and identifies respiratory tract viruses. The conventional tube culture system with multiple cell lines would ordinarily detect RV within 3-30 days. The SVC/IF method using single cell line (MDCK cells) allowed detection of 81.5% of influenza A virus, 72% of parainfluenza virus, 82.6% of respiratory syncytial virus (RSV) and 79.6% of adenovirus in 24 h.     

Protein kinase C activation causes inhibition of Na/K-ATPase activity in Madin-Darby canine kidney epithelial (MDCK) cells

To evaluate the influence of protein kinase C (PKC) activation on Na/K-ATPase activity in MDCK cells, we studied the effect of phorbol myristate acetate (PMA) and two diacylglycerol analogues, oleoylacetylglycerol and dioctanoylglycerol, on the enzyme activity. Na/K-ATPase activity was determined by cytochemistry. PMA induced a time- and dose-dependent inhibition of Na/K-ATPase activity and at 100 ng/ml decreased the enzyme activity by 55% of the initial value. These effects were mimicked by oleoylacetylglycerol and dioctanoylglycerol, and were abolished by two inhibitors of PKC, 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7) and sphingosine. A phorbol ester that does not activate PKC, 4-phorbol 12, 13-didecanoate, did not inhibit Na/ K-ATPase activity. PMA inhibition persisted in the presence of cycloheximide and actinomycin D but not in the presence of amiloride. Dopamine (10 M) inhibition of Na/K-ATPase activity was abolished in a dose-dependent manner by sphingosine. Results suggest that in MDCK cells Na/K-ATPase is an effector protein for PKC and that dopamine inhibition of its activity may be mediated by PKC.     

Comparison of tube cultures of Madin Darby canine kidney cells with shell-vial cultures after low-speed centrifugation for influenza virus isolation

The efficacy of isolation of influenza viruses was compared in two systems--conventional tube cultures of Madin Darby canine kidney cells, and shell-vial cultures after low-speed centrifugation. One hundred and fifty eight throat and nasal swabs were inoculated and cultures screened by hemagglutination on days 1, 2, 3 and 5 post-inoculation. Twenty-four shell-vial cultures (15.2%) and 6 tube cultures (3.8%) were positive for influenza virus type B, indicating that shell-vial cultures with low-speed centrifugation are superior to tube cultures for influenza virus type B isolation (p less than 0.005 by McNemar's test for analysis of matched pairs data).     

Two-dimensional manipulation of differentiated Madin-Darby canine kidney (MDCK) cell sheets: the noninvasive harvest from temperature-responsive culture dishes and transfer to other surfaces

A renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells, adheres, spreads, and proliferates to confluency on our developed temperature-responsive culture dishes grafted with a poly(N-isopropylacrylamide) (PIPAAm) at 37 degrees C. In addition to other cell types, including hepatocytes and endothelial cells, MDCK cell sheets noninvasively were harvested from PIPAAm-grafted dishes merely by reducing the temperature. However, during the early stage of culture (up to 3 weeks), confluent MDCK cell detachment is greatly repressed. In the present study, we succeeded in the rapid harvest of confluent MDCK cell sheets and intact transfer to other culture dishes by utilizing hydrophilically modified poly(vinylidene difluoride) (PVDF) membranes as supporting materials. Immunocytochemistry with anti-beta-catenin antibody revealed that the functional cell-cell junctions were well organized in the transferred MDCK cell sheets. The viability assay showed that the transferred cells were not damaged during the two-dimensional cell-sheet manipulation. By transmission electron microscopy it was confirmed that the harvested MDCK cells retained differentiated phenotypes and had many microvilli and tight junctions at the apical and lateral plasma membranes, respectively. This two-dimensional cell-sheet manipulation technique promises to be useful in tissue engineering as well as in the investigation of epithelial cell sheets.     

Adaptation of a Madin-Darby canine kidney cell line to suspension growth in serum-free media and comparison of its ability to produce avian influenza virus to Vero and BHK21 cell lines

Madin-Darby canine kidney (MDCK) cells are currently considered for influenza vaccine manufacturing. A drawback of these cells is their anchorage dependent growth, which greatly complicates process scale-up. In this paper a novel MDCK cell line (MDCK-SFS) is described that grows efficiently in suspension and retained high expression levels of both α-2,6 and α-2,3 sialic acid receptors, which bind preferably to human and avian influenza viruses, respectively. The production of avian influenza virus by BHK21, Vero and MDCK-SFS cell lines was compared. Although BHK21 cells consisted of two populations, one of which lacks the α-2,3 receptor, they supported the replication of two influenza strains to high titres. However, BHK21 cells are generally not applicable for influenza production since they supported the replication of six further strains poorly. MDCK-SFS cells yielded the highest infectious virus titres and virus genome equivalent concentration for five of the eight influenza strains analyzed and the highest hemagglutination activity for all eight virus strains. Taken together with their suitability for suspension growth this makes the MDCK-SFS cell line potentially useful for large scale influenza virus production.     

Some ultrastructural effects of persistent infections by the rickettsia Coxiella burnetii in mouse L cells and green monkey kidney (Vero) cells.

Mouse fibroblasts (L-929) and Vero (green monkey kidney) cells were infected with the rickettsia Coxiella burnetti, and persistent infections developed and were studied over a 6- to 10-month period. Ultrastructural comparisons were made between the two infected cell types, and both were tested cytochemically for the presence of acid phosphatase, a marker enzyme of lysozymes. Rickettsiae were always observed within vacuoles, and some infected L cells showed flattened endoplasmic reticulum as compared with uninfected cells. Rickettsiae in Vero cells were most often seen in vacuoles containing whorls of membranes ("myelin configurations") which were also seen in uninfected cells. Rickettsiae in Vero cells were pleomorphic, with acid phosphatase reaction product in their periplasmic space. This suggests either rickettsial degradation by lysosomal enzymes which penetrated the cell envelope or a penetration after the rickettsiae were dead. Vacuoles of infected Vero cells showed much more reaction product than that in infected L cells, and most rickettsiae in L cells had a normal appearance and showed no reaction product in their periplasmic space.