Infectious Diseases: Using xCELLigence for Virology Studies

Tim Howell xCELLigence

The RTCA method gave results very similar to the traditional method and was significantly quicker and easier to analyze.David Thirkettle-Watts and Penny Gauci (Australian Department of Defense)
Using xCELLigence for Virology Studies

Electron micrograph of Ebola virus.

Virus infection of a host cell typically includes the selective suppression of host cell functions and redirection of resources towards viral replication and assembly, ultimately leading to host cell lysis.  While host cell rounding, detachment from the plate surface and/or lysis are readily detected by the xCELLigence® RTCA biosensors, more subtle changes in host cell morphology occurring during earlier phases of viral infection are also monitored.

This sensitivity to virus-induced changes in host cell morphology and behavior makes the xCELLigence® technology very well suited for a wide array of virology applications including, but not limited to: differentiating between virus strains/isolates based on the kinetics of replication and cytopathic effect, determining viral titers, determining neutralizing antibody titers, drug screening, gene therapy, and studying virus-host cell interactions using physiologically relevant cell types that cannot typically be used because they aren’t compatible with traditional assay techniques.

[The xCELLigence assay can] provide additional data when compared to classical methods. The system allowed dense real-time data collection over several days, combined with low operative effort, and avoided the danger of potentially missing significant events as may happen in end-point assays. In summary, the presented [xCELLigence-based] methods outmatch end-point assays by observing the cell population throughout the entire experiment while workload and time to result are reduced.Witkowski et al. Biochem Biophys Res Commun. 2010 Oct 8;401(1):37-41.

View of a single E-Plate well.

ACEA’s patented microtiter plates (E-Plates®) contain gold biosensors that are integrated into the bottom surface of each well. In the photo to the right, which is looking down into a single well from a 96-well plate, the array of gold biosensors is seen to cover ~75% of the well bottom’s surface area.

xCELLigence® instruments cause a miniscule electric current to flow between the E-Plate biosensors. Adherent cells act as insulators, impeding this flow of current. The ease with which this current flows is directly dependent upon the number of cells attached to the plate bottom, the size of the cells, and the cell-substrate attachment quality. Adherent cells act as insulators, impeding current flow. Importantly, side-by-side assays run in E-Plates and standard plastic microtiter plates have demonstrated that neither the gold biosensors nor the electronic monitoring have any impact on cell attachment, proliferation, etc.

Within ACEA’s E-Plates, virus-induced changes in cell morphology and attachment strength (hallmarks of a cytopathic effect) are readily detected by changes in the biosensor signal. This principle is represented schematically below, where a single well is shown at two different time points.  Note that, for clarity, only two biosensors are shown in the well bottom.

Citing the fact that traditional plaque assays are labor intensive and time consuming, Reisen and colleagues evaluated the efficacy of xCELLigence Real-Time Cell Analysis (RTCA) for determining virus titers.  Vero cells in suspension were incubated with serial dilutions of a known concentration of West Nile virus (WNV) for 30 minutes at 37°C, followed by immediate addition of the cell/virus suspension to E-Plate® wells and subsequently monitoring impedance in an xCELLigence instrument.  In contrast to uninfected control cells which grew to confluency and maintained a plateaued Cell Index, virus-infected cells displayed a time-dependent decrease in Cell Index down to zero, indicating complete cell lysis (Figure A).  Importantly, the time at which this cytopathic effect occurred correlated extremely well with the known titer of the virus.  This is highlighted by plotting the CIT50 (time required for the Cell Index to decrease by 50%) as a function of virus titer (Figure B).  Using this type of standard curve, it is possible to determine the viral titer in samples of unknown concentration.

Real-time monitoring of West Nile virus cytopathic effect.  (A) The timing of West Nile virus (WNV)-induced cytopathic effect is dependent on virus titer.  After being incubated with serial dilutions of WNV, Vero cells were seeded in the wells of an E-Plate and impedance was monitored for 200 hours.  The thin horizontal line denotes CIT50 (time required for the Cell Index to decrease by 50%).  (B) The time dependence of WNV-induced cytopathic effect correlates with virus titer.  Plotting CIT50 as a function of known virus concentration demonstrates the strength of this correlation.  Data have been adapted from J Virol Methods. 2011 May;173(2):251-8.

Key Benefits of Using xCELLigence for Virology Studies:
  1. Objective quantification: Subjective human observation of cytopathic effects are replaced with objective real-time data.
  2. Reduced workload: Once cells are infected and data acquisition has been initiated, no further involvement is required. Data is continuously recorded for anywhere from minutes to days/weeks. Plaque assays are not needed.
  3. Diverse applications:  As long as the virus being studied has an impact on cell number, cell morphology, or cell-surface attachment strength, it can be observed by impedance-monitoring.

 

Virology Assays Supporting Information:

  • Virology Application:
  1. A New Way to Monitor Virus-Mediated Cytopathogenicity
  • Virology Publications:
  1. Efficient and selective tumor cell lysis and induction of apoptosis in melanoma cells by a conditional replication-competent CD95L adenovirus. Fecker LF, Schmude M, Jost S, Hossini AM, Picó AH, Wang X, Schwarz C, Fechner H, Eberle J.  Exp Dermatol. 2010 Aug;19(8):e56-66.
  2. Cellular impedance measurement as a new tool for poxvirus titration, antibody neutralization testing and evaluation of antiviral substances. Witkowski PT, Schuenadel L, Wiethaus J, Bourquain DR, Kurth A, Nitsche A.  Biochem Biophys Res Commun. 2010 Oct 8;401(1):37-41.
  3. Real-time monitoring of flavivirus induced cytopathogenesis using cell electric impedance technology. Fang, Y., Ye, P., Wang, X., Xu, X., & Reisen, W. Journal of virological methods. 2011;173(2), 251–8.
  4. Fluoroquinolones inhibit human polyomavirus BK (BKV) replication in primary human kidney cells. Sharma BN, Li R, Bernhoff E, Gutteberg TJ, Rinaldo CH.  Antiviral Res. 2011 Oct;92(1):115-23.
  5. Novel, real-time cell analysis for measuring viral cytopathogenesis and the efficacy of neutralizing antibodies to the 2009 influenza A (H1N1) virus. Tian, D., Zhang, W., He, J., Liu, Y., Song, Z., Zhou, Z., Zheng, M., et al. PloS One. 2012;7(2), e31965.
  6. Impact of human adenovirus type 3 dodecahedron on host cells and its potential role in viral infection. Fender P, Hall K, Schoehn G, Blair GE.  J Virol. 2012 May;86(9):5380-5.
  7. Generation and characterization of a Cowpox virus mutant lacking host range factor CP77. Schuenadel L, Tischer BK, Nitsche A.  Virus Res. 2012 Sep;168(1-2):23-32.
  8. Generation of an adenovirus-parvovirus chimera with enhanced oncolytic potential. El-Andaloussi N, Bonifati S, Kaufmann JK, Mailly L, Daeffler L, Deryckère F, Nettelbeck DM, Rommelaere J, Marchini A.  J Virol. 2012 Oct;86(19):10418-31.
  9. The xCELLigence system for real-time and label-free analysis of neuronal and dermal cell response to equine herpesvirus type 1 infection. Golke A, Cymerys J, Słońska A, Dzieciatkowski T, Chmielewska A, Tucholska A, Bańbura MW.  Pol J Vet Sci. 2012;15(1):151-3.
  10. Characteristics of polyomavirus BK (BKPyV) infection in primary human urothelial cells. Li R, Sharma BN, Linder S, Gutteberg TJ, Hirsch HH, Rinaldo CH.  2013 May 25;440(1):41-50.
  11. Real-time cell analysis–a new method for dynamic, quantitative measurement of infectious viruses and antiserum neutralizing activity Teng Z, Kuang X, Wang J, Zhang X. J Virol Methods. 2013 Nov;193(2):364-70.
  12. Primary cultures of murine neurons for studying herpes simplex virus 1 infection and its inhibition by antivirals. Cymerys J, Dzieciątkowski T, Golke A, Słońska A, Majewska A, Krzyżowska M, Bańbura MW.  Acta Virol. 2013;57(3):339-45.
  13. Oncolytic effects of parvovirus H-1 in medulloblastoma are associated with repression of master regulators of early neurogenesis. Lacroix J, Schlund F, Leuchs B, Adolph K, Sturm D, Bender S, Hielscher T, Pfister SM, Witt O, Rommelaere J, Schlehofer JR, Witt H.  Int J Cancer. 2014 Feb 1;134(3):703-16.
  14. An improved method for determining virucidal efficacy of a chemical disinfectant using an electrical impedance assay. Ebersohn K, Coetzee P, Venter EH.  J Virol Methods. 2014 Apr;199:25-8.
  15. Viral replication kinetics and in vitro cytopathogenicity of parental and reassortant strains of bluetongue virus serotype 1, 6 and 8. Coetzee P, Van Vuuren M, Stokstad M, Myrmel M, van Gennip RG, van Rijn PA, Venter EH.  Vet Microbiol. 2014 Jun 25;171(1-2):53-65.
  16. Toll-like receptor-3 is dispensable for the innate microRNA response to West Nile virus (WNV). Chugh PE, Damania BA, Dittmer DP.  PLoS One. 2014 Aug 15;9(8):e104770.
  17. Antiviral effects of artesunate on JC polyomavirus replication in COS-7 cells. Sharma BN, Marschall M, Rinaldo CH.  Antimicrob Agents Chemother. 2014 Nov;58(11):6724-34.
  18. Chemical induction of unfolded protein response enhances cancer cell killing through lytic virus infection. Prasad V, Suomalainen M, Pennauer M, Yakimovich A, Andriasyan V, Hemmi S, Greber UF.  J Virol. 2014 Nov;88(22):13086-98.
  19. Antiviral effects of artesunate on polyomavirus BK replication in primary human kidney cells. Sharma BN, Marschall M, Henriksen S, Rinaldo CH.  Antimicrob Agents Chemother. 2014;58(1):279-89.
  20. RIG-I specifically mediates group II type I IFN activation in nervous necrosis virus infected zebrafish cells. Chen HY, Liu W, Wu SY, Chiou PP, Li YH, Chen YC, Lin GH, Lu MW, Wu JL. Fish Shellfish Immunol. 2015 Apr;43(2):427-35.
  21. Specific nucleotides at the 3′-terminal promoter of viral hemorrhagic septicemia virus are important for virulence in vitro and in vivo. Kim SH, Guo TC, Vakharia VN, Evensen Ø.  2015 Feb;476:226-32.
  22. Brincidofovir (CMX001) inhibits BK polyomavirus replication in primary human urothelial cells. Tylden GD, Hirsch HH, Rinaldo CH.  Antimicrob Agents Chemother. 2015;59(6):3306-16.
  23. Metabolic alteration–Overcoming therapy resistance in gastric cancer via PGK-1 inhibition in a combined therapy with standard chemotherapeutics. Schneider CC, Archid R, Fischer N, Bühler S, Venturelli S, Berger A, Burkard M, Kirschniak A, Bachmann R, Königsrainer A, Glatzle J, Zieker D.  Int J Surg. 2015 Oct;22:92-8.
  24. Combination of the oral histone deacetylase inhibitor resminostat with oncolytic measles vaccine virus as a new option for epi-virotherapeutic treatment of hepatocellular carcinoma. Ruf B, Berchtold S, Venturelli S, Burkard M, Smirnow I, Prenzel T, Henning SW, Lauer UM.  Mol Ther Oncolytics. 2015 Oct 7;2:15019.
  25. Development of a Real-Time Cell Analysing (RTCA) method as a fast and accurate screen for the selection of chikungunya virus replication inhibitors. Marlina S, Shu MH, AbuBakar S, Zandi K.  Parasit Vectors. 2015 Nov 9;8:579.
  26. Real-time replication of swine vesicular disease virus (SVDV) in cell culture systems in vitro. Paprocka G, Kęsy A.  Bull Vet Inst Pulawy. 2015; 59, 457-462.
  27. A generic screening platform for inhibitors of virus induced cell fusion using cellular electrical impedance. Watterson D, Robinson J, Chappell KJ, Butler MS, Edwards DJ, Fry SR, Bermingham IM, Cooper MA, Young PR.  Sci Rep. 2016 Mar 15;6:22791.
  28. Novel Method Based on Real-Time Cell Analysis for Drug Susceptibility Testing of Herpes Simplex Virus and Human Cytomegalovirus. Piret J, Goyette N, Boivin G.  J Clin Microbiol. 2016 Aug;54(8):2120-7.
  29. The Presumed Polyomavirus Viroporin VP4 of Simian Virus 40 or Human BK Polyomavirus Is Not Required for Viral Progeny Release. Henriksen S, Hansen T, Bruun JA, Rinaldo CH.  J Virol. 2016 Oct 28;90(22):10398-10413.
  30. Novel epi-virotherapeutic treatment of pancreatic cancer combining the oral histone deacetylase inhibitor resminostat with oncolytic measles vaccine virus. Ellerhoff TP, Berchtold S, Venturelli S, Burkard M, Smirnow I, Wulff T, Lauer UM.  Int J Oncol. 2016 Nov;49(5):1931-1944.
  31. Oncolytic Group B Adenovirus Enadenotucirev Mediates Non-apoptotic Cell Death with Membrane Disruption and Release of Inflammatory Mediators. Dyer A, Di Y, Calderon H, Illingworth S, Kueberuwa G, Tedcastle A, Jakeman P, Chia SL, Brown A, Silva MA, Barlow D, Beadle J, Hermiston T, Ferguson DJ, Champion B, Fisher KD, Seymour LW.  Mol Ther Oncolytics. 2016 Dec 10;4:18-30.
  32. A Real-Time Cell Analyzing Assay for Identification of Novel Antiviral Compounds against Chikungunya Virus. Zandi K.  Methods Mol Biol. 2016;1426:255-62.
  33. Oncolytic Adenoviral Delivery of an EGFR-Targeting T-cell Engager Improves Antitumor Efficacy. Fajardo CA, Guedan S, Rojas LA, Moreno R, Arias-Badia M, de Sostoa J, June CH, Alemany R.  Cancer Res. 2017 Apr 15;77(8):2052-2063.
  34. Cell Cycle-Dependent Kinase Cdk9 Is a Postexposure Drug Target against Human Adenoviruses. Prasad V, Suomalainen M, Hemmi S, Greber UF.  ACS Infect Dis. 2017 Jun 9;3(6):398-405.