Functional evaluation for adequacy of MDCK-lineage cells in influenza research
BMC Research Notes volume 12, Article number: 101 (2019)
Influenza is an acute respiratory disease caused by the influenza virus which circulates annually in populations of different species. Madin-Darby Canine Kidney (MDCK) is the most widely utilized cell-line for conducting influenza research. However, the infectivity of various influenza strains in MDCK cells is not equivalent and the productivity of viral propagation is also limited.
We tested the functional adequacy of two MDCK-lineage cell lines, conventional MDCK and MDCK/London, were evaluated by assessing their infectivity of different influenza viral strains with focus forming assays and the cellular toxicity caused by influenza infections by lactate dehydrogenase assay. Moreover, the sensitivity of cells in the presence of the antiviral agent ribavirin was assessed by MTT assay. Our results showed that MDCK/London cells efficiently propagate virus across all influenza viruses tested, are comparable to the utility of Mv1Lu cells, and are superior to conventional MDCK cells in replicating virus as indicated by an increase in virus of three to four logs, particularly in H3N2 infection. Also, the MDCK/London cells were more sensitive to the presence of antiviral drug than conventional MDCK cells. In conclusion, MDCK/London cell line could be a better platform for influenza studies and vaccine development.
Influenza virus causes pandemics and seasonal epidemics worldwide, leading to 5–15% of the population becoming infected and 50,000 deaths annually in United States . Frequently complicated by bacterial infection, influenza infection can lead to influenza-associated pneumonia which, in combination, is implicated in up to 8.4% of all reported deaths to the 122 Cities Mortality Reporting System in the United States . Researchers have widely utilized influenza-susceptible cell lines such as MDCK or Vero for pathogenesis investigation as well as vaccine developments, particularly MDCK culture-derived vaccine against influenza has been approved by European Medicines Agency [3, 4]. As such, it is critical to select the cell type utilized for conducting influenza research and to interpret data generated from these various cell types. Currently, several cell lines in addition to MDCK and Vero have been reported to be susceptible to influenza virus infection such as hamster lung (HmLu-1), monkey kidney (JTC-12), human colon intestinal epithelium cell line (CACO-2), and mink lung epithelial cells (Mu1Lv) [5,6,7].
The MDCK/London (MDCK/Ln) cell line is also a suitable substrate to grow and isolate influenza virus and is available for purchase on the website of Influenza Reagent Resource (IRR; FR-58). In 1985, MDCK/London cells were originated and developed from the Common Cold Laboratory in Salisbury, UK. MDCK/Ln cells display enhanced sensitivity to influenza infections [8, 9]. A recent study revealed that the MDCK/Ln has a faster growth rate and reflects the influenza infection more sensitively in regards to PFU, HA, and NA titers compared to MDCK/SIAT1 and conventional MDCK cells . However, a thorough, in-depth, and functional comparison of MDCK/Ln and conventional MDCK cells has yet to be demonstrated. Here, we compare the viral propagation of MDCK-lineage cells with another cell line, Mv1Lu, which has been shown to be susceptible to influenza infections , by a high throughput focus forming assay which has been described previously .
To validate the infectivity of influenza viruses propagated in egg embryos (H1N1, ATCC: VR1520; H3N2, ATCC: VR544; H9N2, ATCC: VR1642), we directly performed plaque assays to titrate these viruses in different cells, including MDCK-lineage cells and Mv1Lu cell. The results in Fig. 1a showed that the viral titers of H1N1, H3N2, and H9N2 in MDCK/Ln cells were higher than those in conventional MDCK cells and comparable to the titers in Mv1Lu cells. Among these influenza strains, H3N2 exhibited the most divergent outcomes in conventional MDCK and MDCK/Ln cells. As such, we selected the H3N2 strain to investigate the productivity of influenza virions in MDCK and MDCK/Ln cells by measuring intracellular and extracellular viral titers over time. In order to assess intracellular and extracellular viral titers, supernatant from both cell lines infected with H3N2 was collected and the infected cells were subsequently sonicated to release intracellular virions followed by titration via plaque assays. As illustrated in Fig. 1b–e, the intracellular viral titers in MDCK cells increased over time, reaching over 80% of total virions by day 3 post-infection but there was less than 20% of total viral titers were released from conventional MDCK cells (Fig. 1b, d), On the contrary, the extracellular viral titers in MDCK/Ln cells were at the range of 70–80% of total virions and the intracellular titers were much lower than extracellular titers (20–30%) (Fig. 1c, e), suggesting that influenza virions could be more readily released from MDCK/Ln cells which may due to more efficient viral package and contributed to the higher titers outside host cells in MDCK/Ln, but not MDCK cells.
Next, we observed the H1N1- and H3N2-infected MDCK and MDCK/Ln cells by confocal microscopy at 48 h-post infection (h.p.i.), visualizing the influenza nuclear protein (NP) with anti-H1N1 (MBS: PAB7123P) and anti-H3N2 (MBS: PAB7124P) antibodies to further examine the infectivity of conventional MDCK and MDCK/Ln cells. As seen in Fig. 2a, the confocal images revealed increased intracellular fluorescence of H1N1 and H3N2 viral proteins in MDCK/Ln cells compared to conventional MDCK cells which was confirmed by corrected total cell fluorescence (CTCF) quantification of the fluorescence intensity of NP in both cell types with ImageJ software (Fig. 2b) . However, there is no such significance also determined by Student’s t test between H1N1 in both cell lines, and these data to some degree corresponded with our previous findings in Fig. 1b, c.
Next, we performed functional assays in order to further characterize MDCK/Ln cells as a model for influenza infection. We evaluated the cytotoxicity resulting from influenza infections by Lactate dehydrogenase (LDH) assays (CytoTox 96®, Promega). The cytotoxicity, as measured by the increase of LDH, in both MDCK and MDCK/Ln cells continually increased following infection with H1N1, H3N2, and influenza B virus (IBV; ATCC: VR1883). However, compared to the gradual increase of LDH concentration in conventional MDCK cells, the LDH concentration in MDCK/Ln cells increased robustly by Day 2 post-infection and remained at higher levels (Fig. 2c, d).
Lastly, we investigated the sensitivity of MDCK-lineage cells in response to an antiviral compound. The broad spectrum and FDA-approved antiviral agent ribavirin (1-beta-d-ribofuranosyl-1,2,4-triazole, Sigma-Aldrich)  was utilized to test whether its antiviral efficacy can be reliably reflected in these MDCK-lineage cell lines. We infected the cells with H1N1, H3N2, or IBV at multiplicity of infection (M.O.I.) of 0.1 along with different concentrations of ribavirin, washed out the viral inoculum and ribavirin with PBS and finally added fresh complete culture medium containing fresh ribavirin for the next 72 h of incubation. The antiviral efficacy was defined as cell viability at end point measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay . The results presented in Fig. 3 suggest that the MDCK/Ln cells exhibit enhanced sensitivity to the ribavirin treatments than conventional MDCK cells in response to these three influenza strains. The 50% of effective concentration (EC50) against H1N1, H3N2, and IBV in MDCK/Ln were 8.623, 4.249, and 27.548 μM, respectively. Conversely, except for the H1N1 infection, the EC50 in conventional MDCK cells were higher (Fig. 3b) or unable to be determined (Fig. 3c). Our findings align with previous observations of differences in the EC50 of ribavirin in H1N1 and H3N2 strains [10, 15, 16]. Importantly, our results demonstrate that MDCK/Ln cells could be a more suitable cell line than for determining antiviral efficacy against influenza infections.
MDCK cells are widely used in influenza research and vaccine development [17, 18], but the viral yield and efficacy of MDCK-derived vaccines are limited [19, 20]. While further investigation of the underlying mechanisms is required, our functional evaluation for MDCK-lineage cells as a model for influenza infections provide an alternative aspect of MDCK/Ln cells. We believe that our research findings regarding MDCK/Ln cells can be used to further the flu vaccine development and influenza-related research.
Dushoff J, Plotkin JB, Viboud C, Earn DJ, Simonsen L. Mortality due to influenza in the United States–an annualized regression approach using multiple-cause mortality data. Am J Epidemiol. 2006;163:181–7.
Arriola CS, Brammer L, Epperson S, Blanton L, Kniss K, Mustaquim D, Steffens C, Dhara R, Leon M, Perez A, et al. Update: influenza activity—United States, September 29, 2013-February 8, 2014. MMWR Morb Mortal Wkly Rep. 2014;63:148–54.
Doroshenko A, Halperin SA. Trivalent MDCK cell culture-derived influenza vaccine Optaflu (Novartis Vaccines). Expert Rev Vaccines. 2009;8:679–88.
Genzel Y, Dietzsch C, Rapp E, Schwarzer J, Reichl U. MDCK and Vero cells for influenza virus vaccine production: a one-to-one comparison up to lab-scale bioreactor cultivation. Appl Microbiol Biotechnol. 2010;88:461–75.
Chiapponi C, Zanni I, Garbarino C, Barigazzi G, Foni E. Comparison of the usefulness of the CACO-2 cell line with standard substrates for isolation of swine influenza A viruses. J Virol Methods. 2010;163:162–5.
Schultz-Cherry S, Dybdahl-Sissoko N, McGregor M, Hinshaw VS. Mink lung epithelial cells: unique cell line that supports influenza A and B virus replication. J Clin Microbiol. 1998;36:3718–20.
Sugimura T, Murakami Y, Ogawa T. The susceptibility of culture cells to avian influenza viruses. J Vet Med Sci. 2000;62:659–60.
Klimov A, Balish A, Veguilla V, Sun H, Schiffer J, Lu X, Katz JM, Hancock K. Influenza virus titration, antigenic characterization, and serological methods for antibody detection. Methods Mol Biol. 2012;865:25–51.
Hossain MJ, Perez S, Guo Z, Chen LM, Donis RO. Establishment and characterization of a Madin-Darby canine kidney reporter cell line for influenza A virus assays. J Clin Microbiol. 2010;48:2515–23.
Lin SC, Kappes MA, Chen MC, Lin CC, Wang TT. Distinct susceptibility and applicability of MDCK derivatives for influenza virus research. PLoS ONE. 2017;12:e0172299.
Kumar P, Bartoszek AE, Moran TM, Gorski J, Bhattacharyya S, Navidad JF, Thakar MS, Malarkannan S. High-throughput detection method for influenza virus. J Vis Exp. 2012. https://doi.org/10.3791/3623.
Parry WL, Hemstreet GP 3rd. Cancer detection by quantitative fluorescence image analysis. J Urol. 1988;139:270–4.
Cameron CE, Castro C. The mechanism of action of ribavirin: lethal mutagenesis of RNA virus genomes mediated by the viral RNA-dependent RNA polymerase. Curr Opin Infect Dis. 2001;14:757–64.
Ferrari M, Fornasiero MC, Isetta AM. MTT colorimetric assay for testing macrophage cytotoxic activity in vitro. J Immunol Methods. 1990;131:165–72.
Shigeta S, Mori S, Baba M, Ito M, Honzumi K, Nakamura K, Oshitani H, Numazaki Y, Matsuda A, Obara T, et al. Antiviral activities of ribavirin, 5-ethynyl-1-beta-d-ribofuranosylimidazole-4-carboxamide, and 6′-(R)-6′-C-methylneplanocin A against several ortho- and paramyxoviruses. Antimicrob Agents Chemother. 1992;36:435–9.
Selvam P, Murugesh N, Chandramohan M, Sidwell RW, Wandersee MK, Smee DF. Anti-influenza virus activities of 4-[(1,2-dihydro-2-oxo-3H-indol-3-ylidene)amino]-N-(4,6-dimethyl-2-pyrimidin-2-yl) benzenesulfonamide and its derivatives. Antivir Chem Chemother. 2006;17:269–74.
Liu B, Hossain MJ, Mori I, Kimura Y. Evaluation of a virus derived from MDCK cells infected persistently with influenza A virus as a potential live-attenuated vaccine candidate in the mouse model. J Med Virol. 2008;80:888–94.
Liu K, Yao Z, Zhang L, Li J, Xing L, Wang X. MDCK cell-cultured influenza virus vaccine protects mice from lethal challenge with different influenza viruses. Appl Microbiol Biotechnol. 2012;94:1173–9.
Abdoli A, Soleimanjahi H, Jamali A, Mehrbod P, Gholami S, Kianmehr Z, Feizi N, Saleh M, Bahrami F, Mokhtari-Azad T, et al. Comparison between MDCK and MDCK-SIAT1 cell lines as preferred host for cell culture-based influenza vaccine production. Biotechnol Lett. 2016;38:941–8.
Oh DY, Barr IG, Mosse JA, Laurie KL. MDCK-SIAT1 cells show improved isolation rates for recent human influenza viruses compared to conventional MDCK cells. J Clin Microbiol. 2008;46:2189–94.
HCT led, drafted and performed the experiments in this study. CWL and SWT collected necessary samples and interpreted the data. CCL performed quantification of images and contributed majorly in manuscript writing and CMC analyzed and evaluated the data in this study for publication in addition to acquire funding management and supervised the whole study. All authors read and approved the final manuscript.
We are grateful for the valuable opinions from Dr. Mathew Matt at Booz Allen Hamilton to improve this manuscript.
The authors declare that they have no competing interests.
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This study was internally funded by Taichung Hospital and National Chung Hsing University (MOE-108-S-0023) in terms of providing experimental supplies and subsidizing labor cost. The funder has no influence on experimental design, data interpretation or manuscript writing, editing, approval, or decision to publish.
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Tsai, HC., Lehman, C.W., Lin, CC. et al. Functional evaluation for adequacy of MDCK-lineage cells in influenza research. BMC Res Notes 12, 101 (2019). https://doi.org/10.1186/s13104-019-4134-2