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.

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