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 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 Comparison of in vitro replication features of H7N3 influenza viruses from wild ducks and turkeys: potential implications for interspecies transmission

2006 ◽  
Vol 87 (1) ◽  
pp. 171-175 ◽  
Author(s):  
Simone Giannecchini ◽  
Laura Campitelli ◽  
Laura Calzoletti ◽  
Maria Alessandra De Marco ◽  
Alberta Azzi ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Influenza Viruses ◽  
Interspecies Transmission ◽  
Madin Darby Canine Kidney ◽  
Binding Properties ◽  
Naturally Occurring ◽  
Darby Canine Kidney ◽  
Canine Kidney

In previous work, it was shown that turkey H7N3 influenza viruses, presumably derived ‘in toto’ from interspecies transmission of duck viruses in Northern Italy, had only 2 aa differences in haemagglutinin and a few amino acid differences as well as a 23 aa deletion in neuraminidase compared with duck viruses. Here, the replication of these duck and turkey viruses in Madin–Darby canine kidney cells was investigated with respect to virus–cell fusion and viral elution from red blood cells. Duck viruses showed similar receptor-binding properties to turkey viruses but possessed a higher pH of fusion activation than the turkey viruses. Conversely, turkey viruses were not able to elute from red blood cells. These data confirm that neuraminidase-stalk deletion impairs the release of virions from cells and also confirm existence of naturally occurring viruses with different pH fusion activities, raising the possibility that these features may play a role in the evolution of influenza viruses in different hosts.

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.

MST3 is involved in ENaC-mediated hypertension

2019 ◽  
Vol 317 (1) ◽  
pp. F30-F42
Author(s):  
Te-Jung Lu ◽  
Wei-Chih Kan ◽  
Sung-Sen Yang ◽  
Si-Tse Jiang ◽  
Sheng-Nan Wu ◽  
...  
Keyword(s):  
Kidney Cells ◽  
Hypertensive Rats ◽  
Madin Darby Canine Kidney ◽  
Spontaneously Hypertensive ◽  
Hypomorphic Mutation ◽  
And Oxidative Stress ◽  
Darby Canine Kidney ◽  
Canine Kidney

Liddle syndrome is an inherited form of human hypertension caused by increasing epithelial Na+ channel (ENaC) expression. Increased Na+ retention through ENaC with subsequent volume expansion causes hypertension. In addition to ENaC, the Na+-K+-Cl− cotransporter (NKCC) and Na+-Cl− symporter (NCC) are responsible for Na+ reabsorption in the kidneys. Several Na+ transporters are evolutionarily regulated by the Ste20 kinase family. Ste20-related proline/alanine-rich kinase and oxidative stress-responsive kinase-1 phosphorylate downstream NKCC2 and NCC to maintain Na+ and blood pressure (BP) homeostasis. Mammalian Ste20 kinase 3 (MST3) is another member of the Ste20 family. We previously reported that reduced MST3 levels were found in the kidneys in spontaneously hypertensive rats and that MST3 was involved in Na+ regulation. To determine whether MST3 is involved in BP stability through Na+ regulation, we generated a MST3 hypomorphic mutation and designated MST3+/− and MST3−/− mice to examine BP and serum Na+ and K+ concentrations. MST3−/− mice exhibited hypernatremia, hypokalemia, and hypertension. The increased ENaC in the kidney played roles in hypernatremia. The reabsorption of more Na+ promoted more K+ secretion in the kidney and caused hypokalemia. The hypernatremia and hypokalemia in MST3−/− mice were significantly reversed by the ENaC inhibitor amiloride, indicating that MST3−/− mice reabsorbed more Na+ through ENaC. Furthermore, Madin-Darby canine kidney cells stably expressing kinase-dead MST3 displayed elevated ENaC currents. Both the in vivo and in vitro results indicated that MST3 maintained Na+ homeostasis through ENaC regulation. We are the first to report that MST3 maintains BP stability through ENaC regulation.

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.

The Influence and Mechanism of Paeoniflorin and Albiflorin on Strychnine and Brucine Transport Through Madin-Darby Canine Kidney/Multidrug Resistance1 Cells In Vitro Model

2020 ◽  
Vol 14 (5) ◽  
pp. 616-623
Author(s):  
Yun-Feng Liu ◽  
Yong-Mei Guan ◽  
Shi-Yu Huang ◽  
Lu Wu ◽  
Wei-Feng Zhu ◽  
...  
Keyword(s):  
In Vitro Model ◽  
Efflux Rate ◽  
Madin Darby Canine Kidney ◽  
Expression Levels ◽  
Transport Studies ◽  
Mdr1 Mrna ◽  
Vitro Model ◽  
Darby Canine Kidney ◽  
Canine Kidney

This research sought to study the influence and potentialmechanism of paeoniflorin and albiflorin on strychnine and brucine transport in MDCK-MDR1 cells regulated by P-gp. Cytotoxicity of drugs was tested by MTT assay, and the transport studies were performed in both directions in MDCK-MDR1 cells. The influence of drugs on P-gp ATPase, and the efflux function of P-gp, the expression levels of P-gp and MDR1 mRNA were also estimated. Strychnine and brucine showed well absorption, and the main transport mechanism might be passive diffusion. Verapamil could significantly decrease the efflux rate (ER) of strychnine and brucine, while the ER of strychnine and brucine was increased significantly when co-administrated with paeoniflorin or albiflorin, indicating that paeoniflorin and albiflorin could promote the efflux of these two alkaloids. Strychnine and brucine could activate the activity of P-gp ATPase, suppress the efflux function of P-gp, but have no significant effect on the expression of P-gp. In addition, strychnine could upregulate the expression of MDR1 mRNA. Paeoniflorin and albiflorin could increase the transmembrane transport of strychnine and brucine mediated by P-gp when co-administrated with strychnine or brucine via stimulating the activity of P-gp ATPase, enhancing the efflux function of P-g, increasing the expression levels of MDR1 mRNA and P-gp.

Transepithelial Resistance and Inulin Permeability as Endpoints in In Vitro Nephrotoxicity Testing

2002 ◽  
Vol 30 (2_suppl) ◽  
pp. 53-59 ◽  
Author(s):  
Tracey Duff ◽  
Simon Carter ◽  
Gemma Feldman ◽  
Gordon McEwan ◽  
Walter Pfaller ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Electrical Resistance ◽  
Mdck Cells ◽  
Ussing Chamber ◽  
Fluorescein Isothiocyanate ◽  
Transepithelial Electrical Resistance ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

Transepithelial electrical resistance (RT) and the flux of fluorescein isothiocyanate (FITC) across Madin Darby canine kidney (MDCK) strain 1 cells and porcine epithelial kidney (LLC-PK1) monolayers were compared between three laboratories for a range of nephrotoxins. The precision of the REMS AutoSampler was similar to that of the Ussing chamber and the ENDOHM® technique, but superior to using chopstick electrodes, for measurements of resistance. The nephrotoxins used were selective for the proximal tubule, and in all cases, LLC-PK1 cells were more sensitive than MDCK cells. In most cases, change in RT was a more sensitive indicator of damage than alterations in FITC flux. The REMS system provides high intra-plate precision for RT measurements and is a higher throughput system, which is applicable to screening for nephrotoxicity in vitro.

scholarly journals Influenza A Virus Inhibits RSV Infection via a Two-Wave Expression of IFIT Proteins

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1171
Author(s):  
Yaron Drori ◽  
Jasmine Jacob-Hirsch ◽  
Rakefet Pando ◽  
Aharona Glatman-Freedman ◽  
Nehemya Friedman ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Influenza Viruses ◽  
Mass Spectrometry Analysis ◽  
Influenza A Viruses ◽  
Rsv Infection ◽  
Syncytial Virus ◽  
Mouse Lungs

Influenza viruses and respiratory syncytial virus (RSV) are respiratory viruses that primarily circulate worldwide during the autumn and winter seasons. Seasonal surveillance has shown that RSV infection generally precedes influenza. However, in the last four winter seasons (2016–2020) an overlap of the morbidity peaks of both viruses was observed in Israel, and was paralleled by significantly lower RSV infection rates. To investigate whether the influenza A virus inhibits RSV, human cervical carcinoma (HEp2) cells or mice were co-infected with influenza A and RSV. Influenza A inhibited RSV growth, both in vitro and in vivo. Mass spectrometry analysis of mouse lungs infected with influenza A identified a two-wave pattern of protein expression upregulation, which included members of the interferon-induced protein with the tetratricopeptide (IFITs) family. Interestingly, in the second wave, influenza A viruses were no longer detectable in mouse lungs. In addition, knockdown and overexpression of IFITs in HEp2 cells affected RSV multiplicity. In conclusion, influenza A infection inhibits RSV infectivity via upregulation of IFIT proteins in a two-wave modality. Understanding the immune system involvement in the interaction between influenza A and RSV viruses will contribute to the development of future treatment strategies against these viruses.

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