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 and dissemination of new virus. While host cell rounding, detachment from the plate surface and/or lysis are readily detected by real-time impedance monitoring, more subtle changes in host cell morphology occurring during earlier phases of viral infection can also be 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: differentiating between virus strains/isolates based on the kinetics of replication and cytopathic effect, determining viral titers, determining neutralizing antibody titers, 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.
APPLICATION HIGHLIGHT: DETERMINING VIRUS TITER
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 wells of ACEA’s electronic microtiter plates (E-Plates®). In contrast to uninfected control cells which grew to confluency and maintained a plateaued Cell Index (CI), virus-infected cells displayed a time-dependent decrease of CI down to zero, indicating complete cell lysis (Figure 1A). 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 CI to decrease by 50%) as a function of virus titer (Figure 1B). Using this type of standard curve, it is possible to determine the viral titer in samples of unknown concentration.
Figure 1. 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.
APPLICATION HIGHLIGHT: EVALUATING FITNESS AND IDENTIFYING DIFFERENT VIRUS STRAINS/ISOLATES
Evaluating the relative fitness of different virus strains/isolates, and determining the identity of a virus isolate can involve a large number of techniques, including: ELISA, PCR, RT-PCR, Western blotting, plaque assays, immunofluorescence, etc. Owing to its ability to kinetically characterize a virus-induced cytopathic effect, xCELLigence real-time cell analysis (RTCA) can be used in place of, or in addition to, some of these traditional assays for characterizing virus fitness and/or identity. In the below example RTCA traces were acquired for Vero cells infected with West Nile virus (WNV) or St. Louis encephalitis virus (SLEV) at the same multiplicity of infection. As seen in Figure 2, the cytopathic effect induced by these two viruses begins at different times post infection and takes different amounts of time to complete (i.e. to reach complete cell lysis). This type of RTCA-based kinetic comparison can be used for assessing the relative fitness/virulence of different virus isolates/strains, or to help identify a virus using RTCA traces from known standards.
Figure 2. Real-time monitoring of cytopathic effects induced by West Nile virus (WNV) and St. Louis encephalitis virus (SLEV). Vero cells in suspension were incubated with either WNV or SLEV at an identical multiplicity of infection. Subsequently the cell/virus suspension was added to a well of an E-Plate. Despite being closely related viruses within the same serocomplex, WNV and SLEV show marked differences in the time at which they begin causing a detectable cytopathic effect: the CIT50 (time required for the Cell Index to decrease by 50%) for SLEV lags behind that of WNV by 55 hours. Moreover, the rate at which these two viruses effect complete cell lysis (i.e. the temporal duration of the cytopathic effect) differs. Data have been adapted from J Virol Methods. 2011 May;173(2):251-8.
APPLICATION HIGHLIGHT: DETERMINING NEUTRALIZING ANTIBODY TITER
Besides being used for therapeutic or prophylactic purposes, neutralizing antibodies also serve as a diagnostic hallmark of infection. One means of determining the presence and/or concentration of antibody in a sample is to evaluate the sample’s ability to neutralize infection by a virus. The automated continuous data acquisition of xCELLigence real-time cell analysis (RTCA), combined with its high reproducibility and minimal hands-on time, make it extremely well suited for this type of assay. In Figure 3A, Vero cell were incubated with a fixed amount of West Nile virus (WNV) that had previously been treated with different dilutions of antisera containing a known concentration of WNV neutralizing antibody. The time at which cells displayed a cytopathic effect (with Cell Index dropping to zero) varied dramatically between the different samples. As expected, at higher dilutions the neutralizing capacity of the antisera decreased. By plotting the CIT50 (time required for the Cell Index to decrease by 50%) as a function of the known neutralizing antibody titer a standard curve can be constructed (Figure 3B). This type of curve can be used for determining neutralizing antibody titer in samples of unknown concentration.
Figure 3. Quantitation of neutralizing antibody titer. (A) Vero cells were treated with West Nile virus (WNV) that had previously been incubated with different dilutions of House finch antisera containing a known WNV neutralizing antibody titer. Whereas the negative control (not exposed to virus) displays a plateaued cell index, the positive control (exposed to virus that was not pretreated with antisera) displays a rapid onset of cytopathic effect (CPE). Pretreatment of WNV with antisera delays the onset of CPE. (B) Correlation between antisera/neutralizing antibody titer and time of CPE onset. Plotting the CIT50 (time required for the Cell Index to decrease by 50%) as a function of antisera/neutralizing antibody titer demonstrates the strength of this correlation. Data have been adapted from J Virol Methods. 2011 May;173(2):251-8.
- Quantify virus titer: An automated, simple, reduced workload alternative to plaque assays.
- Evaluate the fitness of different strains/isolates: The relative fitness of different viruses (natural isolates, engineered mutants, etc.) are readily evaluated using the onset and kinetics of virus-mediated cytopathic effects.
- Determine/confirm virus identity: Real-time kinetic traces of virus-mediated cytopathic effects can be compared to those of characterized viruses to help determine/confirm the identity of a virus.
- Quantify neutralizing antibody titer: Because the time of cytopathic effect onset correlates with neutralizing antibody concentration, standard curves are easy to generate. These can be used for quantifying neutralizing antibody in samples of unknown concentration.
- Rapid assay optimization: Quickly identify the optimal viral titer and assay time point for subsequent screening of inhibitory compounds, neutralizing antibodies and neutralizing serums.