Using xCELLigence for Virology Studies

Virus-induced cytopathic effects (CPEs) can involve changes in host cell morphology, reduced cell-cell and cell-substrate attachment strength, and host cell lysis. By monitoring these changes in real-time, xCELLigence instruments probe viral CPEs in unprecedented detail, and yield quantitative kinetics which can be used for numerous applications.

Electron micrograph of Ebola virus.

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, cell-surface attachment strength, or cell-cell adhesion (i.e. barrier function), it can be observed by xCELLigence.

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.

 

Example data for key virology/vaccine applications:

Virus Titer DeterminationNeutralizing Antibody Detection & QuantificationAnti-viral Drug Studies
Viral Fitness ComparisonsOncolytic VirusesVirucide Efficacy 
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)

Vaccine handbook

Handbook: Explore Viral Cytopathic Effect Assays for Virology and Vaccine Research

  • Viral Titer Determination
  • Detection and Quantification of Neutralizing Antibodies
  • Studying Anti-viral Drugs
  • Testing Virucides
  • Oncolytic Viruses
  • Assessing virus quality/fitness

Download Handbook
Virus webinar

Webinar on Demand: A Groundbreaking Technology for Vaccine Development: New Techniques in Viral CPE Assessment using Real Time Cell Analysis

On Thursday, Nov 29, 2018 at 10am EST, Loic Benair (Sanofi Pasteur, France) and Brandon Lamarche (ACEA Biosciences) will describe new approach aimed at quantifying viral cytopathic effects during vaccine development.

Watch Webinar
App note 18

Application Note 18: A New Way to Monitor Virus-Mediated Cytopathogenicity

A demonstration on the experimental workflow and the power of real-time impedance-based technology to evaluate viral cytopathic effects. 

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[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.

xCELLigence instruments that are compatible with virology/vaccine applications:

Dual PurposeSingle PlateMulti PlateHigh Throughput
RTCA DPRTCA SPRTCA MPRTCA HT
3×16 wells1×96 wells6×96 wellsUp to 4×384 wells

Virology Publications:

  1. Real-time analysis of the cytopathic effect of African swine fever virus. Burmakina G, Bliznetsov K, Malogolovkin A. J Virol Methods. 2018 Jul;257:58-61
  2. 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.
  3. 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.
  4. A Real-Time Cell Analyzing Assay for Identification of Novel Antiviral Compounds against Chikungunya Virus. Zandi K.  Methods Mol Biol. 2016;1426:255-62.
  5. 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.
  6. 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.
  7. 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.
  8. 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.
  9. 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.
  10. 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.
  11. 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.
  12. 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.
  13. 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.
  14. 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.
  15. 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.
  16. 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.
  17. 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.
  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 JC polyomavirus replication in COS-7 cells. Sharma BN, Marschall M, Rinaldo CH.  Antimicrob Agents Chemother. 2014 Nov;58(11):6724-34.
  20. 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.
  21. 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.
  22. 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.
  23. 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.
  24. 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.
  25. 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.
  26. 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.
  27. 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.
  28. 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.
  29. 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.
  30. 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.
  31. 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.
  32. 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.
  33. 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.
  34. 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.
  35. 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.

xCELLigence Research Grant

The research grant winner will be provided access to the xCELLigence Real Time Cell Analysis (RTCA) DP instrument, consumables, and consultation for up to 6 months.  Apply by December 8, 2018.

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