Agilent Virus-Mediated Cytopathogenicity Application Note

Application Note

Cell Analysis

A New Way to Monitor Virus-Mediated

Cytopathogenicity

Author

Brandon Lamarche,
JoyceVelez, and Leyna Zhao
Agilent Technologies, Inc.

Introduction

One of the most important procedures in virology is the measurement of viral
cytopathic effects (CPEs). The plaque assay has long been the gold standard for
quantifying CPEs by providing a direct readout of the number or concentration of
infectious viral particles in a sample. In this technique, a confluent monolayer of host
cells is infected with varying dilutions of the virus and is overlaid with a semisolid
material, such as dilute agarose gel. When an infected cell lyses, the overlay
material prevents the released virions from diffusing through the medium and
infecting distal sites. However, progeny virions can gain access to neighboring cells
in the immediate vicinity. In this manner, infection and lysis spread laterally in two
dimensions and produce a cell-free plaque in the middle of an otherwise confluent
group of cells. Depending on the host cell type and the virus, accurate recognition
and counting of plaques may require staining cells with a dye, such as crystal violet.

A very low multiplicity of infection (MOI) can be achieved using serial dilutions of
virus. Under this condition, each cell that gets infected will be infected by just one
virion. After counting the number of plaques in a well, and accounting for the dilution
factor used in preparing the virus inoculum, the concentration of virus (titer) in the
original sample can be calculated. Titers are usually reported as the number of
plaque forming units (PFU) per unit of volume.

Depending on the virus and host cells being studied, viral plaque formation can take
anywhere from days to weeks to be detectable. A single endpoint plaque assay
provides no information about the onset of CPE or the kinetics of virus-mediated
cytotoxicity. Different cell types and cell densities, as well as viral strains, serotypes,

and mutations can cause plaque formation rates and sizes to vary dramatically.

Thus, the suboptimal selection of a single assay endpoint can result in inaccurate
calculation of viral titer and lytic activity. The definition and manual counting of
plaques by visual inspection can also be highly subjective.

Recent peer-reviewed studies of
oncolytic viruses (Dyer et al., 20172
and Fajardo et al., 20175) and cancer
vaccines (Cross et al., 20151 and
Phametal., 20146) have demonstrated
that the Agilent xCELLigence real-time
cell analysis (RTCA) system is a
powerful tool for evaluating both virus
concentration and cytotoxicity kinetics.
It uses a simple, fast, and reproducible
workflow. Microelectronic biosensors
embedded in the bottom of microplate
wells enable the RTCA assay to offer
dynamic, real-time, label-free, and
noninvasive analysis of cellular events,
such as virus-mediated cytolysis. The
progeny viruses released from a lysed
cell are free to diffuse through the media
and infect distant target cells because an
agarose overlay is not used in the RTCA
procedure. This unhindered spread of
virus throughout the entire well results
in the rapid lysis of all cells, providing a
quantification of viral titer much more
quickly than a plaque assay.

xCELLigence impedance measurements
are automatically recorded at a
user-defined frequency and are plotted
by the xCELLigence software using the
dimensionless parameter known as

CellIndex (CI).

Key benefits of the xCELLigence RTCA
systems for monitoring virus-mediated
cytopathogenicity:

• Label free: No dyes required.

• Fast: Read an entire 96-well plate in
less than 10 seconds.

• Real time: Quantitative monitoring of

both fast (hours) and slow (daysto

weeks) CPE.

• Easy workflow: No gel pouring.
Requires only the addition of virus to
host cells.

• Accurate, precise, and highly
reproducible.

• Automatic data plotting: The
intuitive xCELLigence software
enables easy data display and
objective analysis, precluding the
subjective data vetting that is

common to plaqueassays.

This application note describes the
experimental setup for assessing

vesicular stomatitis virus (VSV)-mediated
cytotoxicity of Vero E6 cells and HEK 293

cells using an xCELLigence instrument.
The protocol shows the identification
of cell proliferation kinetics as well as
the optimal time point for viral infection
with different cell seeding densities.
The assay overcomes many of the
limitations of single-point plaque assays,
and provides direct evidence that RTCA
can offer a comprehensive and reliable
evaluation of viral cytopathogenicity.

Materials and methods

Cells

Cells were cultured in a standard

humidified incubator at 37 °C with
5%CO2 saturation. Vero E6 (obtained

from the ATCC) is an African green
monkey kidney-derived cell line with
deficiency of the type I interferon genes.

HEK 293 (obtained from Microbix

Biosystems) is a human embryonic
kidney cell line with an intact interferon
system. Both adherent cell lines were
maintained in Dulbecco’s Modified Eagle
Medium (DMEM), supplemented with
10% fetal calf serum, 2 mM glutamine,
and 1% penicillin/streptomycin.

Virus

The VSV, Serotype Indiana, was grown
and titrated on Vero E6 cells at 37 °C with

5% CO2.

Cell proliferation assays

For real-time cell analysis, 100 µL of
growth media was added to each well
of the Agilent E-Plate 96 to obtain
background readings. For each cell
type, a sequential 1:1 dilution series,
with seven different cell numbers

ranging from 50,000 to 781cells/well,
were resuspended in 100µL of media,

then seeded into the E-Plate 96. The
E-Plates containing cells were incubated

for 30 minutes at room temperature,

and placed on the RTCA single plate

(SP) station, located in the cell culture

incubator. Cell attachment, spreading,
and proliferation were monitored every

30 minutes using the RTCA SP. Measured

impedance recordings from cells in each
individual well on the E-Plate 96 were
automatically converted to CI values by

the RTCAsoftware.

Assessment of virus-mediated
cytopathogenicity

For viral studies, 25,000 cells/well and
12,500 cells/well of each cell line were
seeded into each well of an E-Plate 96.
When the cells reached confluency
(25,000 cells/well) or were still in the
growth phase (12,500 cells/well),

after 20.5 hours for Vero E6 cells and

68.5hours for HEK 293 cells, they
were infected by VSV. This was done

by removing the E-Plate 96 from the

RTCA SP station and adding 800,000

("high MOI") or 80,000 ("low MOI")

resuspended in 10 μL of growth media to

the wells. As the control, eight wells were

mock-infected by adding 10µL growth

media only. The E-Plate 96 was then
placed immediately back into the RTCA

SP station in the incubator and the CI
values were measured every 15minutes

for up to 190 hours.

2

Results and discussion

A

B

Proliferation curve of Vero E6 cells

Dymanic monitoring of

cellproliferation

To identify the optimal time point for viral
infection, a cell proliferation analysis was

performed with Vero E6 and HEK293
cells. Suitable time points for virus
infection were defined at 20.5hours for
Vero E6 cells and 68.5 hours for HEK
293 cells (Figures 1A and 1B). At these

time points, cells were either in the
growth phase when 12,500 cells had
been used for seeding, or in the early
stationary phase when 25,000 cells
had been used for seeding. Therefore,
viral cytopathogenicity was monitored
in either the growth phase or the early
stationary phase.

15

13

11

9

7

Cell IndexCell Index

5

3

1

-1
0 32 64 96 128 160 192

Time (hours)

Proliferation curve of HEK 293 cells

3.5

2.5

1.5

0.5

-0.5
0 32 64 96 128 160 192

Time (hours)

Figure 1. Dynamic monitoring of cell proliferation. Cells were seeded in the E-Plate 96 and
continuously monitored by measuring CI to identify a suitable time point for addition of virus

(growth or early stationary phase). The adhesion, spreading, and proliferation of (A) Vero E6
cells and (B)HEK 293 cells were dynamically monitored every 30 minutes using the RTCA
SP instrument. Colored curves indicate the different cell numbers seeded per well in an
E-Plate96 (from left to right): red, 50,000; green,25,000; blue, 12,500; magenta, 6,250; cyan,
3,125; orange, 1,562; darkgreen, 781; olive green, medium control (without cells).

50,000 cells
25,000 cells
12,500 cells
6,250 cells
3,125 cells
1,562 cells
781 cells
Medium control

(0 cells)

50,000 cells
25,000 cells
12,500 cells
6,250 cells
3,125 cells
1,562 cells
781 cells
Medium control

(0 cells)

3

VSV cytopathogenicity profile using

A

B

VSV cytopathogenicity profile on Vero E6 cells

Vero E6 cells

At 20.5 hours after seeding (based on the
dynamic monitoring of cell proliferation),

Vero E6 cells either in growth phase or

early stationary phase were infected with

VSV using two different MOIs.

When Vero E6 cells were infected with
VSV during the growth phase, there

was a clear correlation between the
amount of virus used for infection and
the onset of the virus-mediated CPE
(Figure 2). After infection with a low MOI

(80,000PFU VSV), the cells continued

to grow for 15 hours (Figure 2A, blue
curve), similar to mock-infected cells

(Figure2A, green curve). The CI values

then decreased, indicating that the cells

were dying as a consequence of VSV

replication. In contrast, mock-infected
cells continued to grow. At 24 hours after
infection, the CI values had decreased
to 50% of the maximum value (CI50),

then continued to decline to zero,

indicating complete cell death in the
infected culture. In contrast, the CI of

Vero E6 cells infected with a high MOI
(800,000PFU VSV) started to decline

at 4 hours post infection (Figure 2A,

after 11hours.

redcurve), and the CI50 was reached

Very similar results were obtained when
confluent Vero E6 cells were infected

(Figure 2B). The CI50 was reached

at 10hours postinfection (high MOI,
Figure2B, red curve) and 19 hours

postinfection (low MOI, Figure 2B, blue
curve), respectively. Again, complete
death of the infected cultures was
observed, as indicated by the decrease of

CI values tozero.

(12,500 cells/well)

1.6

1.4

1.2

1.0

0.8

0.6

0.4

Normalized Cell IndexNormalized Cell Index

0.2

0

0 11 22 33 44 55 66

Time (hours)

VSV cytopathogenicity profile on Vero E6 cells
(25,000 cells/well)

1.2

1.0

0.8

0.6

0.4

0.2

0

0 11 22 33 44 55 66

Time (hours)

Figure 2. Dynamic monitoring of Vero E6 cells during VSV infection. (A)Normalized Cell
Index values of growing cells, (B) Normalized Cell Index values of confluent cells. The

virus-mediated effect on adhesion, spreading, and proliferation of the cells was monitored

by measuring cell impedance every 15 minutes using the RTCA SP instrument. Time of

addition of virus at 20.5 hours is indicated by the black vertical line. The time point when
the CI value had decreased to 50% of the maximum (CI50) value is indicated by the dotted

orange lines. Green curves: control (no viral infection); bluecurves:80,000 PFU VSV; red
curves:800,000PFU VSV.

Control (no viral
infection)

80,000 PFU VSV
800,000 PFU VSV
CI50

Control (no viral
infection)

80,000 PFU VSV
800,000 PFU VSV
CI50

4

VSV cytopathogenicity profile using

A

B

Time (hours)

HEK 293 cells

Based on the dynamic monitoring of cell

proliferation, HEK 293 cells in the growth

phase and in early stationary phase

were infected with VSV 68.5 hours after
seeding (Figures 3A and 3B).

HEK 293 cells showed a different
response compared to Vero E6 cells
when infected with VSV. HEK 293

cells in the growing phase were much

more sensitive to VSV infection. This

is indicated by the drop in CI values to

HEK 293 cells to VSV infection with

a low MOI may have been due to the
antiviral response mounted by their

interferonsystem.

Confluent cells may also represent

a suboptimal environment for VSV

replication because they have reduced
metabolic activity compared to growing
cells. In line with this hypothesis was
the observation that growing HEK

293 cells are much more sensitive
to VSV, independent of the MOI used
forinfection.

However, the VSV-M protein has been

known to counteract the interferon
system by inhibiting host RNA and
protein synthesis. This contributed
to the shutoff of host-directed gene
expression.4 Therefore, the observed

differences in response to VSV infection
in growing or confluent Vero E6 and
HEK 293 cells and the dependency of
the outcome on cell number (and VSV

MOI) most likely reflected the interplay
of cellular antiviral mechanisms and

viralcountermeasures.

the CI50 value 6 hours after infection

when a high MOI was used (Figure3A,

red curve). Cells infected with a low
MOI reached the CI50 12 hours after

infection (Figure 3A, blue curve). A

completely different result was obtained

when confluent HEK 293 cells were
infected (see Figure 3B). Confluent

cells infected with a high MOI exhibited
a drop in CI values, similar to growing

VSV cytopathogenicity profile on HEK 293 cells
(12,500 cells/well)

2.0

1.5

1.0

Control (no viral
infection)

80,000 PFU VSV
800,000 PFU VSV
CI50

cells (Figure3B, red curve). Cells

infected with a low MOI appear to be

completely resistant to VSV infection,

0.5

Normalized Cell IndexNormalized Cell Index

exhibiting CI values virtually identical to

mock-infected cells (Figure 3B, blue and

green curves). Considering the different

responses to the VSV infection, the main
difference between Vero E6 and HEK293

cells was the ability to produce type I

interferons. Vero E6 cells are devoid of
the interferongenes.

3

As a consequence, Vero E6 cells

could not upregulate the expression

0

65 70 75 80 85 90 95 100 105 110 115

Time (hours)

VSV cytopathogenicity profile on HEK 293 cells
(25,000 cells/well)

1.2

1.0

0.8

0.6

Control (no viral
infection)

80,000 PFU VSV
800,000 PFU VSV
CI50

of interferon-induced antiviral active

proteins, such as MxA and OAS/RNaseL,

in response to viral infections.

In contrast, HEK 293 cells possess an

intact interferon system. During viral

0.4

0.2

0

65 72 79 86 93 100 107

infection, they produced interferons

that activated the JAK/STAT signaling

pathway in an autocrine and paracrine
manner. As a result, the expression of
antiviral active proteins was initiated
and an antiviral state was established.

Theobserved resistance of confluent

Figure 3. Dynamic monitoring of HEK 293 cells during viral infection. (A)Normalized
CellIndex values of growing cells, (B) Normalized Cell Index values of confluent cells. The

virus-mediated effect on adhesion, spreading, and proliferation of the cells was dynamically

monitored every 15 minutes using the RTCA SP instrument. Time of addition of virus at

68.5hours is indicated by the black vertical line. The time point when the CI had decreased to

50% of the maximum value (CI50) is indicated by the dotted orange lines. Green curves show

the control (no viral infection); blue curves: 80,000 PFU VSV; red curves: 800,000 PFU VSV.

5

In contrast to conventional endpoint
assays, real-time cell analysis using
the xCELLigence RTCA system offers
continuous monitoring of virus-host
interactions to better define the
responses where viral or cellular
activities are more dominant.

References

1. Cross, et al. Therapeutic DNA

Vaccination Against Colorectal

Cancer by Targeting the MYB
Oncoprotein. Clin. Transl.
Immunology 2015, 4(1), e30.

2. Dyer, et al. Oncolytic Group B

Adenovirus Enadenotucirev
Mediates Non-apoptotic Cell Death
with Membrane Disruption and
Release of Inflammatory Mediators.

Molecular Therapy: Oncolytics 2017,
4, 18–30.

3. Emeny, J. M.; Morgan, M. J.
Regulation of the Interferon System:
Evidence That Vero Cells Have

a Genetic Defect in Interferon
production. J. Gen. Virol. 1979, 43(1),
247–252.

4. Ferran, M. C.; Lucas-Lenard, J. M.

The Vesicular Stomatitis Virus Matrix

Protein Inhibits Transcription From
the Human Beta Interferon Promoter.
J. Virol. 1997, 71(1), 371–377

5. Fajardo, et al. Oncolytic Adenoviral
Delivery of an EGFR-Targeting

TCell Engager Improves Antitumor

Efficacy, 2017. doi: 10.1158/0008-

5472.CAN-16-1708

6. Pham, et al. A simple in vitro Method
for Evaluating Dendritic Cell-Based

Vaccinations. Onco Targets Ther.

2014, 7, 1455–64.

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