scholarly journals Comparative Susceptibility of Madin–Darby Canine Kidney (MDCK) Derived Cell Lines for Isolation of Swine Origin Influenza A Viruses from Different Clinical Specimens

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2346
Author(s):  
Matthew Suderman ◽  
Mariko Moniwa ◽  
Tamiru N. Alkie ◽  
Davor Ojkic ◽  
Andre Broes ◽  
...  
Keyword(s):  
Cell Lines ◽  
Influenza A ◽  
Mdck Cells ◽  
Influenza A Viruses ◽  
Madin Darby Canine Kidney ◽  
Clinical Specimens ◽  
Oral Fluids ◽  
Nasal Swabs ◽  
Darby Canine Kidney ◽  
Canine Kidney

Madin–Darby canine kidney (MDCK) cells are commonly used for the isolation of mammalian influenza A viruses. The goal of this study was to compare the sensitivity and suitability of the original MDCK cell line in comparison with MDCK-derived cell lines, MDCK.2, MDCK SIAT-1 and MDCK-London for isolation of swine-origin influenza A viruses (IAV-S) from clinical specimens. One-hundred thirty clinical specimens collected from pigs in the form of nasal swabs, lung tissue and oral fluids that were positive by PCR for the presence of IAV-S RNA were inoculated in the cell cultures listed above. MDCK-SIAT1 cells yielded the highest proportion of positive IAV-S isolations from all specimen types. For nasal swabs, 58.62% of the specimens were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (36.21%), and conventional MDCK and MDCK.2 cells (27.5%). For lung specimens, 59.38% were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (40.63%), and conventional MDCK and MDCK.2 cells (18.75–31.25%). Oral fluids yielded the lowest number of positive virus isolation results, but MDCK-SIAT1 cells were still had the highest rate (35%) of IAV-S isolation, whereas the isolation rate in other cells ranged from 5–7.5%. Samples with lower IAV-S PCR cycle threshold (Ct) values were more suitable for culturing and isolation. The isolated IAV-S represented H1N1-β, H1N2-α, H1N1pdm and H3N2 cluster IV and cluster IVB viruses. The result of the current study demonstrated the importance of using the most appropriate MDCK cells when isolating IAV-S from clinical samples.

scholarly journals Generation of a Reassortant Influenza A Subtype H3N2 Virus Expressing Gaussia Luciferase

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 665 ◽  
Author(s):  
Lin Wang ◽  
Qinghua Cui ◽  
Xiujuan Zhao ◽  
Ping Li ◽  
Yanyan Wang ◽  
...  
Keyword(s):  
Influenza A ◽  
Reverse Genetics ◽  
Mdck Cells ◽  
H3n2 Virus ◽  
Influenza A Viruses ◽  
Madin Darby Canine Kidney ◽  
Gaussia Luciferase ◽  
Ns Gene ◽  
Darby Canine Kidney ◽  
Canine Kidney

Reporter influenza A viruses (IAVs) carrying fluorescent or luminescent genes provide a powerful tool for both basic and translational research. Most reporter IAVs are based on the backbone of either subtype H1N1 viruses, A/Puerto Rico/8/1934 (PR8) or A/WSN/1933, but no reporter subtype H3N2 virus is currently available to our knowledge. Since the IAV subtype H3N2 co-circulates with H1N1 among humans causing annual epidemics, a reporter influenza A subtype H3N2 virus would be highly valuable. In this study, the segments of A/Wyoming/3/03 (NY, H3N2) virus encoding hemagglutinin and neuraminidase, respectively, were reassorted with the six internal genes of PR8 where the NS gene was fused with a Gaussia luciferase (Gluc) gene. Using reverse genetics, NY-r19-Gluc, a replication competent reassortant influenza A subtype H3N2 virus expressing reporter Gluc was successfully generated. This reporter virus is stable during replication in Madin-Darby canine kidney (MDCK) cells, and preliminary studies demonstrated it as a useful tool to evaluate antivirals. In addition, NY-r19-Gluc virus will be a powerful tool in other studies including the application of diagnostic and therapeutic antibodies as well as the evaluation of novel vaccines.

scholarly journals Use of influenza A viruses expressing reporter genes to assess the frequency of double infections in vitro

2012 ◽  
Vol 93 (8) ◽  
pp. 1645-1648 ◽  
Author(s):  
R. Bodewes ◽  
N. J. Nieuwkoop ◽  
R. J. Verburgh ◽  
R. A. M. Fouchier ◽  
A. D. M. E. Osterhaus ◽  
...  
Keyword(s):  
Influenza A ◽  
Single Cells ◽  
Reporter Genes ◽  
Influenza Viruses ◽  
Influenza A Viruses ◽  
Double Infection ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

Exchange of gene segments between mammalian and avian influenza A viruses may lead to the emergence of potential pandemic influenza viruses. Since co-infection of single cells with two viruses is a prerequisite for reassortment to take place, we assessed frequencies of double-infection in vitro using influenza A/H5N1 and A/H1N1 viruses expressing the reporter genes eGFP or mCherry. Double-infected A549 and Madin–Darby canine kidney cells were detected by confocal microscopy and flow cytometry.

scholarly journals Mechanism of Antiviral Activity of Nitazoxanide against the Influenza Virus: Effect of Tizoxanide on AdenosineTriphosphate in Influenza-virus Infected Madin Darby Canine Kidney Cells

2021 ◽  
Author(s):  
Jean-Francois Rossignol ◽  
Carl van Baalen ◽  
Aloys Tijsma
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Respiratory Infections ◽  
Mdck Cells ◽  
Dependent Manner ◽  
Madin Darby Canine Kidney ◽  
Atp Production ◽  
Viral Respiratory Infections ◽  
Darby Canine Kidney ◽  
Canine Kidney

Background: Nitazoxanide (NTZ) is a broad-spectrum antiviral undergoing clinical development for treating influenza and other viral respiratory infections such as those caused by rhinovirus/enterovirus and coronavirus including the emerging SARS-CoV-2. Methods: Nitazoxanide is a mild uncoupler of oxidative phosphorylation, which is modulating the ATP production in cells. ATP is an essential component of viral replication, and we have evaluated the effect of tizoxanide (TIZ), the active circulating metabolite of NTZ, on ATP in Madin-Darby canine kidney (MDCK) cells and in MDCK cells infected with influenza A and B viruses. Results: TIZ decreased cellular ATP in a dose-dependent manner in MDCK cells and in MDCK cells infected with influenza A and B viruses. Maximum inhibition of ATP in influenza infected or uninfected MDCK cells reached up to 45% after 6 and 24 hours of exposure to 100 micrometer TIZ. The decrease in cellular ATP did not affect cell viability and was reversible after eliminating TIZ from the culture. Conclusion: The concentrations of TIZ required to decrease cellular ATP levels were similar to those reported to inhibit replication of influenza A and B viruses in our laboratory. A decrease in ATP triggers activation of AMP-activated protein kinase, which is known to suppress the secretion of pro-inflammatory cytokines. Additional studies are warranted to evaluate the effect of TIZ on mitochondrial function.

scholarly journals A Y161F Hemagglutinin Substitution Increases Thermostability and Improves Yields of 2009 H1N1 Influenza A Virus in Cells

2017 ◽  
Vol 92 (2) ◽  
Author(s):  
Feng Wen ◽  
Lei Li ◽  
Nan Zhao ◽  
Meng-Jung Chiang ◽  
Hang Xie ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Influenza A ◽  
Vero Cells ◽  
Influenza Viruses ◽  
High Yield ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

ABSTRACTVaccination is the primary strategy for influenza prevention and control. However, egg-based vaccines, the predominant production platform, have several disadvantages, including the emergence of viral antigenic variants that can be induced during egg passage. These limitations have prompted the development of cell-based vaccines, which themselves are not without issue. Most importantly, vaccine seed viruses often do not grow efficiently in mammalian cell lines. Here we aimed to identify novel high-yield signatures for influenza viruses in continuous Madin-Darby canine kidney (MDCK) and Vero cells. Using influenza A(H1N1)pdm09 virus as the testing platform and an integrating error-prone PCR-based mutagenesis strategy, we identified a Y161F mutation in hemagglutinin (HA) that not only enhanced the infectivity of the resultant virus by more than 300-fold but also increased its thermostability without changing its original antigenic properties. The vaccine produced from the Y161F mutant fully protected mice against lethal challenge with wild-type A(H1N1)pdm09. Compared with A(H1N1)pdm09, the Y161F mutant had significantly higher avidity for avian-like and human-like receptor analogs. Of note, the introduction of the Y161F mutation into HA of seasonal H3N2 influenza A virus (IAV) and canine H3N8 IAV also increased yields and thermostability in MDCK cells and chicken embryotic eggs. Thus, residue F161 plays an important role in determining viral growth and thermostability, which could be harnessed to optimize IAV vaccine seed viruses.IMPORTANCEAlthough a promising complement to current egg-based influenza vaccines, cell-based vaccines have one large challenge: high-yield vaccine seeds for production. In this study, we identified a molecular signature, Y161F, in hemagglutinin (HA) that resulted in increased virus growth in Madin-Darby canine kidney and Vero cells, two cell lines commonly used for influenza vaccine manufacturing. This Y161F mutation not only increased HA thermostability but also enhanced its binding affinity for α2,6- and α2,3-linked Neu5Ac. These results suggest that a vaccine strain bearing the Y161F mutation in HA could potentially increase vaccine yields in mammalian cell culture systems.

Differential sensitivity of Madin-Darby canine kidney (MDCK) cells to epinephrine

2015 ◽  
Vol 20 (5) ◽  
pp. 486-493 ◽  
Author(s):  
P. Muthuraman ◽  
P. C. Nagajyothi ◽  
M. Chandrasekaran ◽  
G. Enkhtaivan ◽  
B. Venkitasamy ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Differential Sensitivity ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

Accumulation of natural and synthetic betaines by a mammalian renal cell line

1996 ◽  
Vol 74 (2) ◽  
pp. 283-287 ◽  
Author(s):  
K. Randall ◽  
M. Lever ◽  
B. A. Peddie ◽  
S. T. Chambers
Keyword(s):  
Osmotic Stress ◽  
Glycine Betaine ◽  
Mdck Cells ◽  
Intracellular Accumulation ◽  
Madin Darby Canine Kidney ◽  
Renal Cell Line ◽  
High Concentrations ◽  
Inhibition Studies ◽  
Darby Canine Kidney ◽  
Canine Kidney

Intracellular accumulation of different betaines was compared in osmotically stressed Madin Darby canine kidney (MDCK) cells to model the betaine accumulation specificity of the mammalian inner medulla and to show how this accumulation differed from that of bacteria. All betaines accumulated less than glycine betaine. Arsenobetaine (the arsenic analogue of glycine betaine) accumulated to 12% of the glycine betaine levels and the sulphur analogue dimethylthetin accumulated to >80%. Most substituted glycine betaine analogues accumulated to 2–5% of intracellular glycine betaine concentrations, however, serine betaine accumulated to <0.5% of glycine betaine levels. Inhibition studies to distinguish the betaine ports were performed by the addition of proline. Butyrobetaine and carnitine accumulation was not proline sensitive, whereas that of omer betaines was. As with glycine betaine, the accumulation of propionobetaine and dimethylthetin was proline sensitive and osmoregulated. Pyridinium betaine was accumulated by both proline-sensitive and -insensitive systems, with a small increase under osmotic stress. High concentrations (10 times that of glycine betaine) of the dietary betaines proline betaine and trigonelline inhibited total betaine accumulation. Because α-substituted betaines are accumulated by bacteria and not by MDCK cells, these betaines may be the basis for design of antimicrobial agents.Key words: MDCK cells, betaine accumulation, osmolytes, betaine analogues.

Damaging effects of high energy shock waves on cultured Madin Darby Canine Kidney (MDCK) cells

1990 ◽  
Vol 18 (4) ◽  
pp. 255-258 ◽  
Author(s):  
W. L. Strohmaier ◽  
K. -H. Bichler ◽  
P. Deetjen ◽  
S. Kleinknecht ◽  
M. Pedro ◽  
...  
Keyword(s):  
Shock Waves ◽  
Mdck Cells ◽  
High Energy ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
Damaging Effects

scholarly journals TGN38 recycles basolaterally in polarized Madin-Darby canine kidney cells.

1994 ◽  
Vol 5 (10) ◽  
pp. 1093-1103 ◽  
Author(s):  
A K Rajasekaran ◽  
J S Humphrey ◽  
M Wagner ◽  
G Miesenböck ◽  
A Le Bivic ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Protein Localization ◽  
Evolutionary Relationship ◽  
Cytoplasmic Domain ◽  
Chimeric Proteins ◽  
Madin Darby Canine Kidney ◽  
Surface Domains ◽  
Darby Canine Kidney ◽  
Canine Kidney

Sorting of newly synthesized plasma membrane proteins to the apical or basolateral surface domains of polarized cells is currently thought to take place within the trans-Golgi network (TGN). To explore the relationship between protein localization to the TGN and sorting to the plasma membrane in polarized epithelial cells, we have expressed constructs encoding the TGN marker, TGN38, in Madin-Darby canine kidney (MDCK) cells. We report that TGN38 is predominantly localized to the TGN of these cells and recycles via the basolateral membrane. Analyses of the distribution of Tac-TGN38 chimeric proteins in MDCK cells suggest that the cytoplasmic domain of TGN38 has information leading to both TGN localization and cycling through the basolateral surface. Mutations of the cytoplasmic domain that disrupt TGN localization also lead to nonpolarized delivery of the chimeric proteins to both surface domains. These results demonstrate an apparent equivalence of basolateral and TGN localization determinants and support an evolutionary relationship between TGN and plasma membrane sorting processes.

Nonmitogenic morphoregulatory action of pp60v-src on multicellular epithelial structures

1987 ◽  
Vol 7 (4) ◽  
pp. 1326-1337
Author(s):  
S L Warren ◽  
W J Nelson
Keyword(s):  
Tyrosine Kinases ◽  
Mdck Cells ◽  
Growth Potential ◽  
Junctional Complex ◽  
Madin Darby Canine Kidney ◽  
Epithelial Monolayers ◽  
Zonula Occludens ◽  
Darby Canine Kidney ◽  
Canine Kidney

Madin-Darby canine kidney (MDCK) cells form polarized, multicellular epithelial structures in vitro. Low-level expression of pp60v-src in MDCK cells elicits plasticity in these multicellular structures. Plasticity was revealed by the displacement of cells from mechanically stressed regions of the epithelial monolayers; however, the two-dimensional relationship between the cells in the remainder of the monolayer was maintained. Electron microscopy of multicellular structures revealed abnormal separation of the lateral membranes of adjacent cells and selective uncoupling of the junctional complex; the zonula adherens was disrupted, but the zonula occludens and desmosomes were retained. Significantly, this result was not accompanied by transformation of the cells, as judged by the absence of anchorage-independent growth potential. These results demonstrate a nonmitogenic biological activity of pp60v-src which is experimentally dissociable from transformation. This morphoregulatory action on higher-order epithelial structures may reflect a function of related cellular tyrosine kinases.

scholarly journals The MAL Proteolipid Is Necessary for Normal Apical Transport and Accurate Sorting of the Influenza Virus Hemagglutinin in Madin-Darby Canine Kidney Cells

1999 ◽  
Vol 145 (1) ◽  
pp. 141-151 ◽  
Author(s):  
Rosa Puertollano ◽  
Fernando Martín-Belmonte ◽  
Jaime Millán ◽  
María del Carmen de Marco ◽  
Juan P. Albar ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Ectopic Expression ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Transport Pathway ◽  
Influenza Virus Hemagglutinin ◽  
Darby Canine Kidney ◽  
Canine Kidney

The MAL (MAL/VIP17) proteolipid is a nonglycosylated integral membrane protein expressed in a restricted pattern of cell types, including T lymphocytes, myelin-forming cells, and polarized epithelial cells. Transport of the influenza virus hemagglutinin (HA) to the apical surface of epithelial Madin-Darby canine kidney (MDCK) cells appears to be mediated by a pathway involving glycolipid- and cholesterol- enriched membranes (GEMs). In MDCK cells, MAL has been proposed previously as being an element of the protein machinery for the GEM-dependent apical transport pathway. Using an antisense oligonucleotide-based strategy and a newly generated monoclonal antibody to canine MAL, herein we have approached the effect of MAL depletion on HA transport in MDCK cells. We have found that MAL depletion diminishes the presence of HA in GEMs, reduces the rate of HA transport to the cell surface, inhibits the delivery of HA to the apical surface, and produces partial missorting of HA to the basolateral membrane. These effects were corrected by ectopic expression of MAL in MDCK cells whose endogenous MAL protein was depleted. Our results indicate that MAL is necessary for both normal apical transport and accurate sorting of HA.

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