Real-time replication of swine vesicular disease virus (SVDV) in cell culture systems in vitro

Abstract

A swine vesicular disease virus (SVDV) replication assay in IB-RS-2, SK-6, and PK-15 cell cultures was performed using the xCELLigence system. The cell status was monitored by impedance measurement, expressed as cell index (CI). Proliferation of particular cells was examined at the beginning of the study. The cells exhibited the ability to form a monolayer, and the CI values increased with the cell culture growth. After about 23 h and while still in the growth phase, the cells were infected with decimal virus dilutions (10

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Bull Vet Inst Pulawy 59, 457-462, 2015
DOI:10.1515/bvip-2015-0067
DE GRUYTER
OPEN
DE
G
Real-time replication
of swine vesicular disease virus (SVDV)
in cell culture systems
in vitro
Grażyna Paprocka, Andrzej Kęsy
Department of Foot-and-Mouth Disease,
National Veterinary Research Institute, 98-320 Zduńska Wola, Poland
grazyna.paprocka@piwzp.pl
Received: August 26, 2015 Accepted: November 26, 2015
Abstract
A swine vesicular disease virus (SVDV) replication assay in IB-RS-2, SK-6, and PK-15 cell cultures was performed
using the xCELLigence system. The cell status was monitored by impedance measurement, expressed as cell index (CI).
Proliferation of particular cells was examined at the beginning of the study. The cells exhibited the ability to form
a monolayer, and the CI values increased with the cell culture growth. After about 23 h and while still in the growth phase,
the cells were infected with decimal virus dilutions (10-1–10-6) containing from 100 000 to 1 median tissue culture infectious
doses (TCID50). SVDV replication in cell cultures induced a change in cell index; together with the occurrence of cytopathic
effect (CPE), the CI values declined. A significant correlation between the concentration of the virus used and CPE
occurrence was found. The results also enabled determination of cell sensitivity to SVDV infection. The highest sensitivity
was exhibited by IB-RS-2, followed by SK-6. To conclude, the xCELLigence System was used effectively and evaluated as
being an efficient tool for CPE detection and SVDV replication analysis in cell cultures. Compared to the standard method , it
enabled a more precise assessment of viral replication based on the quantitative CI measurement, providing additional current
information.
Keywords: swine vesicular disease, impedance, cell index, cytopathic effect.
Introduction
Swine vesicular disease (SVD) affects all breeds
of domestic pigs and wild boars. It is listed by the
World Organisation for Animal Health (OIE) as
subject to notification and official control.
The aetiological agent of the disease is a virus
(swine vesicular disease virus – SVDV) belonging to
the family Picornaviridae in the Enterovirus species,
and antigenically related to human pathogens
Coxsackie B5 and A16. Only one serotype of the
virus is known, but antigenically different isolates
occur, varying in virulence and tropism (1, 6).
The disease was first recognised in Italy in 1966,
where it is still detected, whereas in Poland the
outbreaks were observed in the 1970s. The disease is
characterised by vesicles developing on the limbs and,
to a lesser extent, the oral mucous membranes; it is
clinically indistinguishable from other diseases with
vesicular lesions during their course, including the
most serious and the most easily spread of them, foot-
and-mouth disease. The presence of the virus or
specific neutralising antibodies constitutes the basis
for the detection of the disease. The methods
recommended by the OIE are an isolation test and
indirect sandwich ELISA for the detection and
identification of the virus, PCR for the detection of its
genetic material, and monoclonal antibody
competitive ELISA (MAC-ELISA) and a standard
neutralisation test for serology.
The methods involving the use of sensitive cells
are among those with the highest sensitivity and
specificity (7, 8). In the case of viral infections,
studies involving cell cultures are considered the gold
standard in diagnosis. SVDV proliferates in primary
and secondary swine cell cultures and in the
continuous cell lines IB-RS-2, SK-6, and PK-15. This
is a cytopathogenic virus, whose replication in the
above-mentioned cultures leads to morphological
changes as a cytopathic effect (CPE) visible in
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microscopy. CPE is a basis for studies using cell
cultures. A novel method for the real-time assay of
in vitro cells is real-time cell analysis (RTCA) (4, 5,
9, 10, 11, 14), also applied to diagnosis of viral
diseases (2, 3, 12, 13), and it creates a possibility for
improvement in that area. The aim of this experiment
was to evaluate the applicability of RTCA in CPE
detection in SVDV-infected cell cultures.
Material and Methods
Cell lines. The porcine kidney cell lines IB-RS-2,
SK-6, and PK-15 were propagated in 75 cm2 bottles in
Eagle’s minimal essential medium (MEM) (Sigma-
Aldrich, USA), supplemented with 10% foetal bovine
serum (FBS) (Gibco, part of Thermo Scientific, USA)
and antibiotics (100 U/mL penicillin and 100 μg/mL
streptomycin, Sigma-Aldrich, USA). IB-RS-2 cells
were obtained from the Pirbright Institute (UK),
whereas SK-6 and PK-15 originated from the
Department of Swine Diseases at the National
Veterinary Research Institute in Pulawy, Poland.
Virus adaptation to cell cultures. The cultures
were infected with a laboratory-developed strain of
SVDV. They were passaged two or three times until
they showed 90%–100% CPE.
Impedance and cell index measurement. The
measurements were performed using the xCELLigence
system (Roche Applied Science, ACEA Biosciences,
USA) which consisted of the RTCA dual-plate (DP)
station for three 16-well culture plates (E-plate 16 and
E-plate View 16), combined with an analyser and a
computer with integrated software. Specially designed
plates with a glass bottom are coated with gold
microelectrodes, which take up approximately 80% of
the plate well surface. The system enables dynamic
monitoring of adherent cells in real time through
a quantitative impedance measurement in each plate
well, expressed as a cell index (CI) value. CI is a non-
dimensional parameter which provides information on
the biological status of the cells, their changing
numbers, morphology, and adhesion; it rises with an
increasing number of cells or stronger adhesion, and
declines with their decreasing number or weaker
adhesion.
Prior to the assay, the RTCA DP station was
placed in an incubator at 37ºC (±1oC) with
a controlled atmosphere of 5% CO2 (±0.5%), and
connected to a computer located outside the incubator.
Next, 100 μL of growth medium was added to each
plate well and the plates were placed inside the station
in order to measure the background that is the cell-
free signal. One plate was designated for each type of
cells. After the measurements, 100 μL of a particular
cell suspension was added, at about 50 000 cells per
well. The plates were left at room temperature for
30 min and then placed inside the station, still
remaining in the incubator. CI measurement was
registered every 30 min. After about 23 h of cell
growth, the cultures on plates were inoculated with
a virus adapted to a particular cell line, in tenfold
increasing dilutions of 10-1–10-6 (100 000–1 median
tissue culture infectious doses (TCID50)), at 50 μL per
well. Two wells were designated for each dilution,
whereas the control consisted of uninfected cultures.
After the plates were placed in the RTCA DP station,
the CI measurements were continued.
Simultaneously, a standard assay was performed
following the infection of cell cultures in Nunc 96-
well flat bottom plates (Nunc, part of Thermo
Scientific, USA). The cytopathic effect was
microscopically inspected only during working hours.
Results
The obtained results are presented in Figs 1–3.
Fig. 1 shows the dynamics of SVDV replication in the
IB-RS-2 cell culture. The virus in doses of 100 000,
10 000, 1 000, 100, 10, and 1 TCID50 induced
a decline of the CI values after 4, 5, 8½, 11, 12, and
18 h respectively. In the SK-6 cell culture infected
with the virus in doses of 100 000, 10 000, 1 000, 100,
and 10 TCID50, the CI values declined after 5, 6, 8,
12, and 19 h respectively (Fig. 2). Finally, in the
PK-15 cell culture, the CI values declined only after
inoculation with the virus in doses of 100 000, 10 000,
and 1 000 TCID50, after 6, 7, and 9½ h respectively
(Fig. 3). In the SK-6 culture infected with a 1 TCID50
dose of the virus and in the PK-15 culture inoculated
with doses 100, 10, and 1 TCID50, CPE was not
detected. These cells continued to grow, and in the
case of PK-15 the CI values were almost identical to
the control. The cytopathic effect in IB-RS-2 cell
culture on an E-plate View 16 and on a standard plate
is shown in Figs 4, 5.
Discussion
The study demonstrated the efficacy of
xCELLigence for CPE detection in IB-RS-2, SK-6,
and PK-15 cell cultures infected with SVDV. First,
cell proliferation was assayed. The cells showed the
ability to form monolayers, and CI values increased
due to culture growth. Eagle’s MEM as used was
appropriate for impedance measurement, from which
data were collected in real time, in both numerical
and graphic form. In microscopic examination,
satisfactory culture growth in the plate wells (E-plate
View 16) was observed, comparable with that
obtained on the plate used in the standard method.
The cells were infected with SVDV doses ranging
from 100 000 to 1 TCID50 while still in the growth
phase. Monitoring of the infection kinetics followed,
based on the changes of the cell index value. Viral
replication induced the occurrence of CPE in the cell
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G. Paprocka, A. Kęsy/Bull Vet Inst Pulawy/59 (2015) 457-462 459
culture. Each curve was characterised by a cell growth
phase correlated with an increase in CI value, and
a consecutive CI drop, reflecting cytopathic effect.
These were dependent on the dose of the virus used,
which indicates the possibility of SVDV titration
using the xCELLigence system. A clear correlation
between the TICD50 doses of the virus used for
infection and the occurrence of CPE was observed. In
the cultures infected with higher SVDV
concentrations, the drop in CI value due to viral
replication occurred earlier and continued until
reaching zero, which indicated the death of the
infected cell culture. When infected with lower
concentrations, CI values dropped later or the cells
continued to grow, similarly to uninfected cells. The
ultimate results of CPE detection in cell cultures using
the xCELLigence system and by means of the
standard method were comparable, which implies that
this new technique is appropriate for measuring the
changes in cellular parameters during infection. This
study has also enabled researchers to assay sensitivity
of the cells used to SVDV infection; the highest
sensitivity was exhibited by IB-RS-2, followed by
SK-6. Few authors have published the results
of studies similar in scope. Interesting data
emerged from CPE monitoring in Vero cell cultures
infected with Saint Louis encephalitis virus and West
Nile virus, both of the Flaviviridae family (2).
Furthermore, the study conducted by Golke et al. (3)
showed the efficacy of the new technique for
monitoring a primary murine neuron culture and
equine dermal (ED) cell lines infected with equine
herpesvirus type 1 from the Herpesviridae family.
The system of real-time cell analysis was also used
for monitoring CPE in a Madin–Darby canine kidney
(MDCK) cell culture infected with strains of A/H1N1
influenza virus (12), as well as for titration of the
virus from the Poxviridae family in a human embryo
kidney 293 cell culture (13).
Summing up, the xCELLigence system was used
effectively and assessed as being an efficient tool for
CPE detection and analysis of SVDV replication in
IB-RS-2, SK-6, and PK-15 cell cultures. Compared to
the standard method, it enabled a more precise
assessment of viral replication in real time based on
the CI measurement and provided additional current
information on the predominance of either viral or
cellular activity in cell–virus interaction. A further
advantage was the possibility of constant control over
the quality of cells during the whole course of the
study. With a view to improving and expanding
laboratory diagnostics of swine vesicular disease,
further investigations using the xCELLigence system
based on more comprehensive material are justified,
with regard to detection of the virus through isolation
and viral antibodies by seroneutralisation.
Fig. 1. SVDV replication in IB-RS-2 cell culture. Curves 1, 2, 3, 4, 5, and 6 match the virus doses of 100 000, 10 000, 1 000, 100, 10,
and 1 TCID50. K – control, uninfected cell culture
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460 G. Paprocka, A. Kęsy/Bull Vet Inst Pulawy/59 (2015) 457-462
Fig. 2. SVDV replication in SK-6 cell culture. Curves 1, 2, 3, 4, 5, and 6 match the virus doses of 100 000, 10 000, 1 000, 100, 10, and 1 TCID50.
K – control, uninfected cell culture
Fig. 3. SVDV replication in PK-15 cell culture. Curves 1, 2, 3, 4, 5, and 6 match the virus doses of 100 000, 10 000, 1 000, 100, 10, and
1 TCID50. K – control, uninfected cell culture
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G. Paprocka, A. Kęsy/Bull Vet Inst Pulawy/59 (2015) 457-462 461
Fig. 4. Cytopathic effect in IB-RS-2 cell culture on an SVDV-infected E-plate View 16. A – control, uninfected cell culture; B – cell culture at
24 h after 1 TCID50 infection
Fig. 5. Cytopathic effect in IB-RS-2 cell culture on an SVDV-infected 96-well flat bottom plate. A – control, uninfected cell culture; B – cell
culture at 24 h after 1 TCID50 infection
Conflict of Interests Statement: The authors declare
that there is no conflict of interests regarding the
publication of this article.
Acknowledgements: The authors would like to thank
Roche Diagnostics Polska and Biotech Poland for
providing access to the xCELLigence system.
References
1. Escribano-Romero E., Jimenez-Clavero M.A., Ley V.: Swine
vesicular disease virus. Pathology of the disease and molecular
characteristics of the virion. Anim Health Res Rev 2000, 1,
119–126.
2. Fang Y., Ye P., Wang X., Xu X., Reisen W.: Real-time
monitoring of flavivirus induced cytopathogenesis using cell
electric impedance technology. J Virol Methods 2011, 173,
251–258.
3. Golke A., Cymerys J., Słońska A., Dzieciątkowski T.,
Chmielewska A., Tucholska A., Bańbura M.W.: The
xCELLigence system for real-time and label-free analysis of
neuronal and dermal cell response to equine herpesvirus type 1
infection. Pol J Vet Sci 2012, 15, 151–153.
4. Huang B., Jin D., Zhang J., Sun J.Y., Wang X., Stiles J., Xu X.,
Kamboj M., Babady N.E., Tang Y.W.: Real-time cellular
analysis coupled with a specimen enrichment accurately detects
and quantifies Clostridium difficile toxins in stool. J Clin
Microbiol 2014, 52, 1105–1111.
5. Irelan J.T., Wu M.-J., Morgan J., Ke N., Xi B., Wang X., Xu X.,
Abassi Y.A.: Rapid and quantitative assessment of cell quality,
identity, and functionality for cell-based assays using real-time
cellular analysis. J Biomol Screen 2011, 16, 313–322.
6. Lin F., Kitching R.P.: Swine Vesicular Disease: An Overview.
Vet J 2000, 160, 192–201.
7. Paprocka G.: Detection of swine vesicular disease virus in
biological materials. Med Weter 2010, 2, 118–120.
8. Paprocka G., Niedbalski W., Kęsy A., Fitzner A.: Detection of
foot-and-mouth disease virus in biological samples – recent
diagnostics possibilities. Bull Vet Inst Pulawy 2010, 54,
467–472.
9. Ryder A.B., Huang Y., Li H., Zheng M., Wang X., Stratton
C.W., Xu X., Tang Y.W.: Assessment of Clostridium difficile
infections by quantitative detection of tcdB toxin by use of
a real-time cell analysis system. J Clin Microbiol 2010, 48,
4129–4134.
10. Slanina H., König A., Claus H., Frosch M., Schubert-
Unkmeir A.: Real-time impedance analysis of host cell response
to meningococcal infection. J Microbiol Methods 2011, 84,
101–108.
Unauthenticated
Download Date | 1/15/16 11:04 PM

462 G. Paprocka, A. Kęsy/Bull Vet Inst Pulawy/59 (2015) 457-462
11. Solly K., Wang X., Xu X., Strulovici B., Zheng W.: Application
of real-time cell electronic sensing (RT-CES) technology to cell-
based assays. Assay Drug Dev Technol 2004, 2, 363–372.
12. Tian D., Zhang W., He J., Liu Y., Song Z., Zhou Z., Zheng M.,
Hu Y.: Novel, real-time cell analysis for measuring viral
cytopathogenesis and the efficacy of neutralizing antibodies to
the 2009 influenza A (H1N1) virus. PLoS ONE 2012, 7, e31965.
13. Witkowski P.T., Schuenadel L., Wiethaus J., Bourquain D.R.,
Kurth A., Nitsche A.: Cellular impedance measurement as a new
tool for poxvirus titration, antibody neutralization testing and
evaluation of antiviral substances. Biochem Biophys Res
Commun 2010, 401, 37–41.
14. Teng Z., Kuang X., Wang J., Zhang X.: Real-time cell analysis –
a new method for dynamic, quantitative measurement of
infectious viruses and antiserum neutralizing activity. J Virol
Methods 2013, 193, 364–370.
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