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An improved method for determining virucidal efficacy of a chemical disinfectant using an electrical impedance assay
Journal of Virological Methods 199 (2014) 25–28
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
An improved method for determining virucidal efficacy of a chemical disinfectant using an electrical impedance assay Karen Ebersohn ∗ , Peter Coetzee, E.H. Venter Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa
a b s t r a c t Article history: Received 20 June 2013 Received in revised form 7 November 2013 Accepted 11 December 2013 Available online 3 January 2014 Keywords: Electrical impedance assay Virucide efficacy Infectious bursal disease virus Cytopathic effect
A major problem with the testing of virucidal efficacy using current protocols is that scoring of virusinduced cytopathic effect (CPE) is dependent on subjective visual interpretation using light microscopy. The current report details the use of an electrical impedance assay (xCELLigence, ACEA Biosciences) for its utility in virucidal efficacy testing. In this study, the xCELLigence system was used in a procedure developed from guidelines given by the Deutsche Vereiniging zur Bekämpfung der Viruskrankheiten (DVV) (German Association for the Control of Virus Diseases) in order to demonstrate the inactivation of infectious bursal disease virus using a commercial virucide. Although the modified DVV assay using the xCELLigence system yielded identical results (i.e. a 5-log 10 reduction in viral infectivity) as the traditional DVV assay, the system allows virucidal efficacy and cytotoxicity to be measured in a more precise and reproducible fashion. © 2014 Elsevier B.V. All rights reserved.
Regular cleaning and disinfection to reduce the level of bacterial and viral pathogens is essential in the domestic farming environment and veterinary and medical practices (Kassaify et al., 2007). There are currently two methods that are widely used for testing the efficacy of virucidal compounds, i.e. the viral carrier and suspension tests. The viral carrier test attempts to determine viral inactivation on hard surfaces (instrumentation, hands, or other surfaces) following contact exposure to a virucidal compound of interest, whereas in the suspension test, virus is incubated together with the virucidal compound in a liquid suspension. Both methods are dependent on evaluating viral inactivation by observing the development of viral cytopathic effect (CPE) in permissible cell cultures (Bellamy, 1995). In Europe two methods are available for viral suspension testing, the German DVV (Deutsche Vereiniging zur Bekämpfung der Viruskrankheiten) (Blümel et al., 2009) and French AFNOR (Agence Francaise de Normalisation) (AFNOR, 1989) tests. Both methods assume virucidal efficacy if a 4-log 10 reduction in viral titre occurs following compound exposure, although this reduction in viral infectivity cannot always be demonstrated due to the cytotoxicity of many compounds in cell cultures at lower dilutions (Bellamy, 1995). A major problem with virucidal efficacy testing is that there is currently no broadly accepted universal standard, which makes it difficult to compare results between laboratories. In particular, the
∗ Corresponding author. Tel.: +27 012 529 8338; fax: +27 012 529 8312. E-mail address: [email protected] (K. Ebersohn). 0166-0934/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2013.12.023
scoring of CPE is based on the visual interpretation of cell culture deterioration using light microscopy, which is subjective. Recently a new method has been developed for measuring virus-induced CPE by using an electrical impedance system (xCELLigence, System, ACEA Biosciences) (Spiegel, 2009). This system uses specially coated cell culture plates, called E-plates, in which the wells are coated at the bottom with gold micro-electrode sensor arrays. The system can be used to visualize virus-induced CPE in a software assisted manner, in real-time, by measuring changes in electrical impedance (CI, cell index value) in infected cell monolayers. The current report describes the use of the xCELLigence system in a modified DVV test, in order to evaluate the inactivation of infectious bursal disease virus (IBDV, genus Avibirnavirus, family Birnaviridae) using a commercial virucide. The virucide testing was carried out in Vero monolayer cultures (Vero 76, American Type Culture Collection, Manassa, USA). Vero cells were propagated in Minimal Essential Medium (MEM) supplemented with 5% gamma-irradiated fetal calf serum (Highveld Biological, Edenvale, South Africa), 10% tryptose phosphate broth (v/v) and 1 mg/L gentamicin (Highveld Biological). Cells were maintained at 37 ◦ C and 98% humidity until 80% confluent. In order to prepare a virus stock, a field isolate of IBDV was sequentially passaged four times in 80% confluent Vero cells using 75 cm2 flasks (Corning). Once 100% CPE was observed at the final passage, the cell culture flask was frozen at −80 ◦ C, thawed and the viruscontaining cell culture supernatant separated from cell debris by centrifugation at 2000 × g for 2 min. The virus containing cell culture supernatant was subsequently titrated in Vero cell cultures and viral titres expressed as log10 TCID50 /mL of cell culture medium
26
K. Ebersohn et al. / Journal of Virological Methods 199 (2014) 25–28
Fig. 1. (A) Virucide mixture. (B) Virus control mixture. (C) Virucidal test mixture. Cytopathic effect of (A) different virucide control dilutions, (B) virus dilutions, and (C) virus–virucide mixture dilutions on Vero cells (DVV assay) as scored using the xCELLigence system. Each curve is composed of CI values from three independent replicates. Cell index values were measured at 30 min intervals following mixture addition. A CI value of zero indicates complete detachment of cells from the bottom of the cell culture wells. Cytotoxicity of the virucide control mixture (A) was measured only from 10−1 to 10−4 dilutions. Standard deviations are indicated for each reading/curve by error bars.
(Kärber, 1931). The virucidal compound was obtained from a commercial supplier and tested at the recommended active dilution and contact period (20 min) according to the manufacturers’ specifications. For comparative purposes, the DVV assay was conducted using both a conventional method, in which CPE was scored visually, as well as by using the xCELLigence system. In order to prepare the assays, 96-well E-plates were seeded with 80 l of a 480 000 cells/mL suspension (38 400 cells) and allowed to grow to confluence overnight. Cytotoxicity control, virus control and virucidal test mixtures were prepared. The first concentration of the virucidal compound used was at 1.25 times the recommended test concentration. The virus control mixture consisted of 6-log10 TCID50 /mL virus in MEM. The virucidal test mixture consisted of a 1:5 dilution of virus in the virucidal compound. All
mixtures were incubated at room temperature for 20 min (contact time), before being serially diluted (eight tenfold dilutions). The 96-well E-plates were divided into three equal parts for the three mixtures and 200 l of the appropriate dilutions of the mixtures added at approximately 24 h post cell seeding. Three wells were used for each dilution. Cell culture and cell free medium controls were included. The plates were incubated for seven days at 37 ◦ C in an atmosphere containing 5% CO2 and CPE scored either by visual interpretation using light microscopy at 7 days post mixture addition (conventional DVV assay) or cell index (CI) values measured every 30 min on the xCELLigence system (RTCA-SP-96). For the xCELLigence assay, a reduction in cell index value of 50% (CI50 ) was used as an estimate of the rate of induction of cell death. Virucidal activity was calculated by comparing the titre of the virus in
K. Ebersohn et al. / Journal of Virological Methods 199 (2014) 25–28
Dilution
Toxicity test
Virus control
Virucidal test
10−1 10−2 10−3 10−4 10−5 10−6 10−7 10−8
+ − − − − − NA NA
+ + + + + + − −
+ − − − − − − −
the absence of the disinfectant with the infectivity obtained in its presence. A 4-log 10 reduction in viral titre was used as criteria to indicate compound efficacy. Visual interpretation of cell culture wells for the conventional DVV assay (Table 1) indicated that the virucidal compound was cytotoxic for Vero cell cultures at the 10−1 dilution (characterized by the rapid degradation of cell culture monolayer integrity, i.e. cell rounding and detachment), whereas no CPE was visible for the 10−2 to 10−4 dilutions. The virus control mixture induced CPE from 10−2 to 10−6 dilutions (characterized by a slow onset CPE from 2 days post infection), whereas the mixture of virus and virucide compound demonstrated cytotoxicity at the 10−1 dilution. A 5-log 10 reduction in viral infectivity was therefore recorded for the 20 min contact period. No CPE was visible in the cell culture control wells. The CPE profiles as measured with the xCELLigence system for the virucide control, virus control and virucidal test mixtures are shown in Fig. 1A–C, whereas the results are summarized in Table 2. Cytotoxicity was characterized by the rapid and precipitous decline in cell culture monolayer integrity following virucide addition [CI50 = 26.2 (±0.3)], whereas for viral infection CI50 was delayed with cell death appearing to be more gradual [CI50 = 50.2 (±0.3)–110.7 (±4.1)]. Examination of the CPE profiles generated with the xCELLigence system indicated that the virucidal compound (Fig. 1A) was cytotoxic for Vero cell cultures at the 10−1 dilution; whereas no cytotoxicity was detected at the 10−2 to 10−4 dilutions (i.e. cell index profiles for each dilution is similar to the cell control). The virus control mixture was capable of inducing CPE in Vero cell cultures from 10−1 to 10−6 dilutions (Fig. 1B). A linear relationship existed between the CI50 values (Fig. 2) between the 10−1 and 10−6 dilutions (correlation coefficient R2 = 0.966). The results indicate cytotoxicity of the virucidal compound (Fig. 1C) at the 10−1 dilution, whereas viral infectivity was neutralized at the 10−2 to 10−6 dilutions. Cumulatively the assay results indicate that similar to the conventional DVV assay, a 5-log 10 reduction in viral infectivity occurred for the 20 min contact period. In the current report a newly developed electrical impedance assay technology (xCELLigence, ACEA Biosciences) was assessed for its utility in virucide efficacy suspension testing. Results obtained using the xCELLigence system gave an identical reduction in viral Table 2 Time to cell index 50 (CI50 ) of the DVV assay as scored using the xCELLigence system. The time to CI50 was determined as the time point at which the CI values for each cell index curve had decreased to 50% of the maximum value. Dilution
Toxicity test
Virus control
Virucidal test
10−1 10−2 10−3 10−4 10−5 10−6 10−7 10−8
26.2 (±0.3) NA NA NA NA NA NA NA
50.2 (±0.3) 57.2 (±0.3) 66.2 (±0.4) 77.2 (±0.3) 90.7 (±0.4) 110.7 (±4.1) NA NA
26.2 (±0.3) NA NA NA NA NA NA NA
120
110.7
100
Time in hours
Table 1 Results of the DVV assay in which conventional visual-based CPE scoring was used (7 days post mixture addition). The development of CPE is indicated as follows: no CPE present (−), non-applicable (NA), and CPE present (+).
27
90.7
80
77.2
66.2
60 50.2
40
R² = 0.9661
57.2 CI50 values
20 0 -1
-2
-3
-4
-5
-6
Virus diluon
Fig. 2. Linear regression analysis demonstrating the correlation between cell index 50 (CI50 ) values and virus control mixture dilutions. The goodness of fit line is indicated in black and the coefficient of correlation (R2 ) is equal to 0.966.
infectivity to the conventional DVV assay (i.e. 5-log 10 reduction in IBDV infectivity). The xCELLigence system provides several advantages over conventional visual-based scoring systems in cell culture-based virucide testing protocols. An initial evaluation of different cell numbers in a cell proliferation assay can be used to establish reproducible maximum cell index values for each individual cell culture well on a 96-well plate, as long as culture conditions are kept constant (e.g. medium composition and incubation conditions) and cells demonstrate the same characteristics (i.e. identical cell lines, passage level, size, number, strength of adherence, virus susceptibility, etc.). This allows for both inter- and intra-laboratory/test standardization, as it can be ensured that the same number of cells are used and that the phenotypic characteristics of cell lines as well as culture conditions are comparable between different test batches. The assay also allows for the precise measurement of CI50 values that can be used to evaluate the accuracy of virus and/or virucide dilution. In the current report, it was demonstrated that a linear relationship existed in CI50 value over the 10−1 to 10−6 dilution range of the virus control mixture. The linear relationship between virus dose and CI50 value could be affected for example by incorrect dilution of virus control and/or virucide test mixtures, and could therefore be used as an additional criterion to evaluate test accuracy and repeatability. In conclusion the xCELLigence system offers an alternative method for evaluating virus-induced CPE in virucidal efficacy testing and yields comparable results to the standard DVV assay prepared using conventional cell culture methods. The xCELLigence system however provides specific software generated data parameters (CPE profiles and CI50 values) that may assist with test standardization. The xCELLigence system is available in either DP (16 well × 3) or SP (96 well × 1) formats and consists of a plate reader (station), analyzer and control unit. The price of the RTCASP and -DP system varies from 50 000.00 to 80 000 Euros, whereas the price of single 16- and 96-well plates varies from 50.00 to 150.00 Euros (approx. 1.5–3 Euros/well). The RTCA plate reader is situated within a CO2 incubator and the system can easily be integrated into standard cell culture laboratories using adherent cell lines and standard media. Additional validation of the method using the comprehensive criteria of either the DVV and/or AFNOR guidelines is required before the technology can be implemented for routine virucide efficacy testing. References AFNOR, 1989. Determination of virucidal activity – viruses of vertebrates (T 72-180). In: Antiseptique set désinfectants. AFNOR, Paris. Bellamy, K., 1995. A review of the test methods used to establish virucidal activity. Journal of Hospital Infection 30 (Suppl.), 389–396.
28
K. Ebersohn et al. / Journal of Virological Methods 199 (2014) 25–28
Blümel, J., Glebe, D., Neumann-Haefelin, D., Rabenau, H.F., Rapp, I., von Rheinbaben, F., Ruf, B., Sauerbrei, A., Schwebke, I., Steinmann, J., Willkommen, H., Wolff, M.H., Wutzler, P., 2009. Guideline of Deutsche Vereinigung zur Bekämpfung der Viruskrankheiten e.V. (DVV; German Association for the Control of Virus Diseases) and Robert Koch-Institute (RKI; German Federal Health Authority) for testing the virucidal efficacy of chemical disinfectants in the human medical area. Hygiene & Medicine 34 (7/8), 293–299. Kärber, G., 1931. Beitrazurkovecktiven Behandlungpharmakologischer Reihenversucbe. Archiv der Experimentellen und Pathologischen Pharmakologie 162, 480–483.
Kassaify, R.G., El Hakim, R.G., Rayya, E.G., Shaib, H.A., Barbour, E.K., 2007. Preliminary study on the efficacy and safety of eight individual and blended disinfectants against poultry and dairy indicator organisms. Veterinaria Italiana 43, 821–830. Spiegel, M., 2009. Real-time and dynamic monitoring of virus mediated cytopathogenicity. Biochemica 3 http://www.roche-appliedscience.com/ wcsstore/RASCatalogAssetStore/Articles/BIOCHEMICA 3 09 p15-17.pdf (accessed 06.07.13).
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
An improved method for determining virucidal efficacy of a chemical disinfectant using an electrical impedance assay Karen Ebersohn ∗ , Peter Coetzee, E.H. Venter Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa
a b s t r a c t Article history: Received 20 June 2013 Received in revised form 7 November 2013 Accepted 11 December 2013 Available online 3 January 2014 Keywords: Electrical impedance assay Virucide efficacy Infectious bursal disease virus Cytopathic effect
A major problem with the testing of virucidal efficacy using current protocols is that scoring of virusinduced cytopathic effect (CPE) is dependent on subjective visual interpretation using light microscopy. The current report details the use of an electrical impedance assay (xCELLigence, ACEA Biosciences) for its utility in virucidal efficacy testing. In this study, the xCELLigence system was used in a procedure developed from guidelines given by the Deutsche Vereiniging zur Bekämpfung der Viruskrankheiten (DVV) (German Association for the Control of Virus Diseases) in order to demonstrate the inactivation of infectious bursal disease virus using a commercial virucide. Although the modified DVV assay using the xCELLigence system yielded identical results (i.e. a 5-log 10 reduction in viral infectivity) as the traditional DVV assay, the system allows virucidal efficacy and cytotoxicity to be measured in a more precise and reproducible fashion. © 2014 Elsevier B.V. All rights reserved.
Regular cleaning and disinfection to reduce the level of bacterial and viral pathogens is essential in the domestic farming environment and veterinary and medical practices (Kassaify et al., 2007). There are currently two methods that are widely used for testing the efficacy of virucidal compounds, i.e. the viral carrier and suspension tests. The viral carrier test attempts to determine viral inactivation on hard surfaces (instrumentation, hands, or other surfaces) following contact exposure to a virucidal compound of interest, whereas in the suspension test, virus is incubated together with the virucidal compound in a liquid suspension. Both methods are dependent on evaluating viral inactivation by observing the development of viral cytopathic effect (CPE) in permissible cell cultures (Bellamy, 1995). In Europe two methods are available for viral suspension testing, the German DVV (Deutsche Vereiniging zur Bekämpfung der Viruskrankheiten) (Blümel et al., 2009) and French AFNOR (Agence Francaise de Normalisation) (AFNOR, 1989) tests. Both methods assume virucidal efficacy if a 4-log 10 reduction in viral titre occurs following compound exposure, although this reduction in viral infectivity cannot always be demonstrated due to the cytotoxicity of many compounds in cell cultures at lower dilutions (Bellamy, 1995). A major problem with virucidal efficacy testing is that there is currently no broadly accepted universal standard, which makes it difficult to compare results between laboratories. In particular, the
∗ Corresponding author. Tel.: +27 012 529 8338; fax: +27 012 529 8312. E-mail address: [email protected] (K. Ebersohn). 0166-0934/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2013.12.023
scoring of CPE is based on the visual interpretation of cell culture deterioration using light microscopy, which is subjective. Recently a new method has been developed for measuring virus-induced CPE by using an electrical impedance system (xCELLigence, System, ACEA Biosciences) (Spiegel, 2009). This system uses specially coated cell culture plates, called E-plates, in which the wells are coated at the bottom with gold micro-electrode sensor arrays. The system can be used to visualize virus-induced CPE in a software assisted manner, in real-time, by measuring changes in electrical impedance (CI, cell index value) in infected cell monolayers. The current report describes the use of the xCELLigence system in a modified DVV test, in order to evaluate the inactivation of infectious bursal disease virus (IBDV, genus Avibirnavirus, family Birnaviridae) using a commercial virucide. The virucide testing was carried out in Vero monolayer cultures (Vero 76, American Type Culture Collection, Manassa, USA). Vero cells were propagated in Minimal Essential Medium (MEM) supplemented with 5% gamma-irradiated fetal calf serum (Highveld Biological, Edenvale, South Africa), 10% tryptose phosphate broth (v/v) and 1 mg/L gentamicin (Highveld Biological). Cells were maintained at 37 ◦ C and 98% humidity until 80% confluent. In order to prepare a virus stock, a field isolate of IBDV was sequentially passaged four times in 80% confluent Vero cells using 75 cm2 flasks (Corning). Once 100% CPE was observed at the final passage, the cell culture flask was frozen at −80 ◦ C, thawed and the viruscontaining cell culture supernatant separated from cell debris by centrifugation at 2000 × g for 2 min. The virus containing cell culture supernatant was subsequently titrated in Vero cell cultures and viral titres expressed as log10 TCID50 /mL of cell culture medium
26
K. Ebersohn et al. / Journal of Virological Methods 199 (2014) 25–28
Fig. 1. (A) Virucide mixture. (B) Virus control mixture. (C) Virucidal test mixture. Cytopathic effect of (A) different virucide control dilutions, (B) virus dilutions, and (C) virus–virucide mixture dilutions on Vero cells (DVV assay) as scored using the xCELLigence system. Each curve is composed of CI values from three independent replicates. Cell index values were measured at 30 min intervals following mixture addition. A CI value of zero indicates complete detachment of cells from the bottom of the cell culture wells. Cytotoxicity of the virucide control mixture (A) was measured only from 10−1 to 10−4 dilutions. Standard deviations are indicated for each reading/curve by error bars.
(Kärber, 1931). The virucidal compound was obtained from a commercial supplier and tested at the recommended active dilution and contact period (20 min) according to the manufacturers’ specifications. For comparative purposes, the DVV assay was conducted using both a conventional method, in which CPE was scored visually, as well as by using the xCELLigence system. In order to prepare the assays, 96-well E-plates were seeded with 80 l of a 480 000 cells/mL suspension (38 400 cells) and allowed to grow to confluence overnight. Cytotoxicity control, virus control and virucidal test mixtures were prepared. The first concentration of the virucidal compound used was at 1.25 times the recommended test concentration. The virus control mixture consisted of 6-log10 TCID50 /mL virus in MEM. The virucidal test mixture consisted of a 1:5 dilution of virus in the virucidal compound. All
mixtures were incubated at room temperature for 20 min (contact time), before being serially diluted (eight tenfold dilutions). The 96-well E-plates were divided into three equal parts for the three mixtures and 200 l of the appropriate dilutions of the mixtures added at approximately 24 h post cell seeding. Three wells were used for each dilution. Cell culture and cell free medium controls were included. The plates were incubated for seven days at 37 ◦ C in an atmosphere containing 5% CO2 and CPE scored either by visual interpretation using light microscopy at 7 days post mixture addition (conventional DVV assay) or cell index (CI) values measured every 30 min on the xCELLigence system (RTCA-SP-96). For the xCELLigence assay, a reduction in cell index value of 50% (CI50 ) was used as an estimate of the rate of induction of cell death. Virucidal activity was calculated by comparing the titre of the virus in
K. Ebersohn et al. / Journal of Virological Methods 199 (2014) 25–28
Dilution
Toxicity test
Virus control
Virucidal test
10−1 10−2 10−3 10−4 10−5 10−6 10−7 10−8
+ − − − − − NA NA
+ + + + + + − −
+ − − − − − − −
the absence of the disinfectant with the infectivity obtained in its presence. A 4-log 10 reduction in viral titre was used as criteria to indicate compound efficacy. Visual interpretation of cell culture wells for the conventional DVV assay (Table 1) indicated that the virucidal compound was cytotoxic for Vero cell cultures at the 10−1 dilution (characterized by the rapid degradation of cell culture monolayer integrity, i.e. cell rounding and detachment), whereas no CPE was visible for the 10−2 to 10−4 dilutions. The virus control mixture induced CPE from 10−2 to 10−6 dilutions (characterized by a slow onset CPE from 2 days post infection), whereas the mixture of virus and virucide compound demonstrated cytotoxicity at the 10−1 dilution. A 5-log 10 reduction in viral infectivity was therefore recorded for the 20 min contact period. No CPE was visible in the cell culture control wells. The CPE profiles as measured with the xCELLigence system for the virucide control, virus control and virucidal test mixtures are shown in Fig. 1A–C, whereas the results are summarized in Table 2. Cytotoxicity was characterized by the rapid and precipitous decline in cell culture monolayer integrity following virucide addition [CI50 = 26.2 (±0.3)], whereas for viral infection CI50 was delayed with cell death appearing to be more gradual [CI50 = 50.2 (±0.3)–110.7 (±4.1)]. Examination of the CPE profiles generated with the xCELLigence system indicated that the virucidal compound (Fig. 1A) was cytotoxic for Vero cell cultures at the 10−1 dilution; whereas no cytotoxicity was detected at the 10−2 to 10−4 dilutions (i.e. cell index profiles for each dilution is similar to the cell control). The virus control mixture was capable of inducing CPE in Vero cell cultures from 10−1 to 10−6 dilutions (Fig. 1B). A linear relationship existed between the CI50 values (Fig. 2) between the 10−1 and 10−6 dilutions (correlation coefficient R2 = 0.966). The results indicate cytotoxicity of the virucidal compound (Fig. 1C) at the 10−1 dilution, whereas viral infectivity was neutralized at the 10−2 to 10−6 dilutions. Cumulatively the assay results indicate that similar to the conventional DVV assay, a 5-log 10 reduction in viral infectivity occurred for the 20 min contact period. In the current report a newly developed electrical impedance assay technology (xCELLigence, ACEA Biosciences) was assessed for its utility in virucide efficacy suspension testing. Results obtained using the xCELLigence system gave an identical reduction in viral Table 2 Time to cell index 50 (CI50 ) of the DVV assay as scored using the xCELLigence system. The time to CI50 was determined as the time point at which the CI values for each cell index curve had decreased to 50% of the maximum value. Dilution
Toxicity test
Virus control
Virucidal test
10−1 10−2 10−3 10−4 10−5 10−6 10−7 10−8
26.2 (±0.3) NA NA NA NA NA NA NA
50.2 (±0.3) 57.2 (±0.3) 66.2 (±0.4) 77.2 (±0.3) 90.7 (±0.4) 110.7 (±4.1) NA NA
26.2 (±0.3) NA NA NA NA NA NA NA
120
110.7
100
Time in hours
Table 1 Results of the DVV assay in which conventional visual-based CPE scoring was used (7 days post mixture addition). The development of CPE is indicated as follows: no CPE present (−), non-applicable (NA), and CPE present (+).
27
90.7
80
77.2
66.2
60 50.2
40
R² = 0.9661
57.2 CI50 values
20 0 -1
-2
-3
-4
-5
-6
Virus diluon
Fig. 2. Linear regression analysis demonstrating the correlation between cell index 50 (CI50 ) values and virus control mixture dilutions. The goodness of fit line is indicated in black and the coefficient of correlation (R2 ) is equal to 0.966.
infectivity to the conventional DVV assay (i.e. 5-log 10 reduction in IBDV infectivity). The xCELLigence system provides several advantages over conventional visual-based scoring systems in cell culture-based virucide testing protocols. An initial evaluation of different cell numbers in a cell proliferation assay can be used to establish reproducible maximum cell index values for each individual cell culture well on a 96-well plate, as long as culture conditions are kept constant (e.g. medium composition and incubation conditions) and cells demonstrate the same characteristics (i.e. identical cell lines, passage level, size, number, strength of adherence, virus susceptibility, etc.). This allows for both inter- and intra-laboratory/test standardization, as it can be ensured that the same number of cells are used and that the phenotypic characteristics of cell lines as well as culture conditions are comparable between different test batches. The assay also allows for the precise measurement of CI50 values that can be used to evaluate the accuracy of virus and/or virucide dilution. In the current report, it was demonstrated that a linear relationship existed in CI50 value over the 10−1 to 10−6 dilution range of the virus control mixture. The linear relationship between virus dose and CI50 value could be affected for example by incorrect dilution of virus control and/or virucide test mixtures, and could therefore be used as an additional criterion to evaluate test accuracy and repeatability. In conclusion the xCELLigence system offers an alternative method for evaluating virus-induced CPE in virucidal efficacy testing and yields comparable results to the standard DVV assay prepared using conventional cell culture methods. The xCELLigence system however provides specific software generated data parameters (CPE profiles and CI50 values) that may assist with test standardization. The xCELLigence system is available in either DP (16 well × 3) or SP (96 well × 1) formats and consists of a plate reader (station), analyzer and control unit. The price of the RTCASP and -DP system varies from 50 000.00 to 80 000 Euros, whereas the price of single 16- and 96-well plates varies from 50.00 to 150.00 Euros (approx. 1.5–3 Euros/well). The RTCA plate reader is situated within a CO2 incubator and the system can easily be integrated into standard cell culture laboratories using adherent cell lines and standard media. Additional validation of the method using the comprehensive criteria of either the DVV and/or AFNOR guidelines is required before the technology can be implemented for routine virucide efficacy testing. References AFNOR, 1989. Determination of virucidal activity – viruses of vertebrates (T 72-180). In: Antiseptique set désinfectants. AFNOR, Paris. Bellamy, K., 1995. A review of the test methods used to establish virucidal activity. Journal of Hospital Infection 30 (Suppl.), 389–396.
28
K. Ebersohn et al. / Journal of Virological Methods 199 (2014) 25–28
Blümel, J., Glebe, D., Neumann-Haefelin, D., Rabenau, H.F., Rapp, I., von Rheinbaben, F., Ruf, B., Sauerbrei, A., Schwebke, I., Steinmann, J., Willkommen, H., Wolff, M.H., Wutzler, P., 2009. Guideline of Deutsche Vereinigung zur Bekämpfung der Viruskrankheiten e.V. (DVV; German Association for the Control of Virus Diseases) and Robert Koch-Institute (RKI; German Federal Health Authority) for testing the virucidal efficacy of chemical disinfectants in the human medical area. Hygiene & Medicine 34 (7/8), 293–299. Kärber, G., 1931. Beitrazurkovecktiven Behandlungpharmakologischer Reihenversucbe. Archiv der Experimentellen und Pathologischen Pharmakologie 162, 480–483.
Kassaify, R.G., El Hakim, R.G., Rayya, E.G., Shaib, H.A., Barbour, E.K., 2007. Preliminary study on the efficacy and safety of eight individual and blended disinfectants against poultry and dairy indicator organisms. Veterinaria Italiana 43, 821–830. Spiegel, M., 2009. Real-time and dynamic monitoring of virus mediated cytopathogenicity. Biochemica 3 http://www.roche-appliedscience.com/ wcsstore/RASCatalogAssetStore/Articles/BIOCHEMICA 3 09 p15-17.pdf (accessed 06.07.13).