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Journal of Virological Methods 187 (2013) 26–31
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Journal of Virological Methods
journal homepage: www.elsevier.com/locate/jviromet
Application of the pseudo-plaque assay for detection and titration of
Crimean-Congo hemorrhagic fever virus
Engin Berber a , Nurettin Canakoglu a , Mustafa D. Yoruk a , Sukru Tonbak a , Munir Aktas b ,
Mustafa Ertek c , Yusuf Bolat a , Ahmet Kalkan d , Aykut Ozdarendeli a,e,∗
a
Department of Virology, College of Veterinary Medicine, Firat University, Elazig, Turkey
Department of Parasitology, College of Veterinary Medicine, Firat University, Elazig, Turkey
c
Refik Saydam National Public Health Agency, Ankara, Turkey
d
Department of Infectious Diseases and Clinical Microbiology, Medical Faculty, Karadeniz Technical University, Trabzon, Turkey
e
Department of Microbiology, Medical Faculty, Erciyes University, Kayseri, Turkey
b
a b s t r a c t
Article history:
Received 24 February 2012
Received in revised form 21 July 2012
Accepted 24 July 2012
Available online 10 August 2012
Keywords:
Titration
Detection
Crimean-Congo hemorrhagic fever
Turkey-Kelkit06
Pseudo-plaque assay
A pseudo-plaque assay was developed for detection and quantitation of Crimean-Congo hemorrhagic
fever virus Turkey-Kelkit06. Enzyme-catalyzed color development of infected cells probed with antiCrimean-Congo hemorrhagic fever virus antibodies was used for determining the titer of Crimean-Congo
hemorrhagic fever Turkey-Kelkit06 and for its detection in samples from persons infected with the
Crimean-Congo hemorrhagic fever virus. The pseudo-plaque assay accuracy was confirmed by comparing pseudo-plaque assay titers with fluorescent immunofocus assay and focus formation assay titers
using three stocks of virus. No significant difference in virus titers of Crimean-Congo hemorrhagic fever
Turkey-Kelkit06 among the three methods was observed. The pseudo-plaque assay is more sensitive than
the fluorescent immunofocus assay for detecting the virus in primary isolates of Crimean-Congo hemorrhagic fever virus collected from humans, but no difference in sensitivity between the two methods was
observed in the cell-adapted strain of Crimean-Congo hemorrhagic fever Turkey-Kelkit06. The pseudoplaque assay is suitable for titration of Crimean-Congo hemorrhagic fever Turkey-Kelkit06, which does
not develop plaques, suggesting it may also be suitable for the detection of other viruses.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
Crimean-Congo hemorrhagic fever virus is a Nairovirus of the
family Bunyaviridae that causes hemorrhage and severe illness in
humans with high mortality rates (Ergonul, 2006; Whitehouse,
2004). The virus is tick-borne, transmitted by the bite of infected
ticks to livestock and humans or by exposure to the tissues or blood
of infected animals (Hoogstraal, 1979). Since Crimean-Congo hemorrhagic fever was first identified in the Crimea and the former
Belgian Congo, the disease has been reported in widespread areas
of Africa, the Middle East, and Eurasia (Ergonul, 2006; Maltezou
et al., 2010; Papa et al., 2002). A large Crimean-Congo hemorrhagic fever outbreak occurred in Turkey in 2002 (Karti et al.,
2004). The numbers of Crimean-Congo hemorrhagic fever cases
∗ Corresponding author at: Department of Microbiology, Medical Faculty, Erciyes
University, Kayseri, Turkey. Tel.: +90 0352 2076666x23387;
fax: +90 0352 437 52 85.
E-mail addresses: [email protected], [email protected]
(A. Ozdarendeli).
0166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jviromet.2012.07.025
have gradually increased in Turkey making the virus a public health
concern (Ergonul, 2006; Ozdarendeli et al., 2010).
Virus detection is the primary method of diagnosis of
early stage disease. Diagnostic assays for acute Crimean-Congo
hemorrhagic fever infection include virus culture, immunohistochemistry, antigen-detection enzyme immunoassay (EIA), and
reverse transcription-PCR (RT-PCR) (Burt et al., 1998; Donets et al.,
1982; Ozdarendeli et al., 2008; Saijo et al., 2002b). Although RTPCR is easy, rapid, and the most sensitive method of detection of
Crimean-Congo hemorrhagic fever virus, it only confirms the presence of the viral genome and does not provide information on the
presence of infectious virions. Currently, plaque assays and fluorescent focus assays are used for measuring virus infectivity of
Crimean-Congo hemorrhagic fever virus in vitro. Alternative methods for measuring virus infectivity of Crimean-Congo hemorrhagic
fever virus have not been considered.
In the present study, a pseudo-plaque assay based on enzymecatalyzed color development of infected cells probed with
anti-Crimean-Congo hemorrhagic fever virus antibodies was used
for determining the titer of Crimean-Congo hemorrhagic fever virus
Turkey-Kelkit06 and for detection of the virus in clinical samples
collected from Crimean-Congo hemorrhagic fever human cases.
E. Berber et al. / Journal of Virological Methods 187 (2013) 26–31
The results obtained by pseudo-plaque assay were compared with
a fluorescence immunofocus assay and a focus formation assay.
2. Materials and methods
2.1. Cells, virus and antibodies
Vero E6 cells (African green monkey kidney) obtained from ATCC
(CRL 1586) and SW-13 cells (adrenocortical carcinoma, Leibovitz
et al., 1973) obtained from the Refik Saydam National Public Health
Agency (RSPHA, Turkey) were maintained in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% heat-inactivated
fetal bovine serum (FBS), 100 mM l-glutamine, 50 U/ml penicillin,
50 ␮g/ml streptomycin (Sigma–Aldrich, Germany).
Crimean-Congo hemorrhagic fever virus Turkey-Kelkit06 strain
was isolated in 2006 from the blood of a patient from the Kelkit
Valley region of Turkey (Tonbak et al., 2006). Crimean-Congo hemorrhagic fever virus Turkey-Kelkit06 was passaged 3 times by
intracerebral inoculations of 2–3 days old suckling mice. The mice
were euthanized 5 days post-infection (PI), and their brains homogenized to 10% (w/v) with DMEM and clarified by centrifugation at
10,000 × g for 15 min. Aliquots were stored at −80 ◦ C. CrimeanCongo hemorrhagic fever virus stocks were prepared on Vero E6
and SW-13 cells by infection of T175 cell culture flasks with a 1:100
dilution of the parent virus stock. Supernatants were collected 4
days PI, cleared of cell debris by centrifugation at 500 × g for 20 min,
at 4 ◦ C, and aliquots were stored at −80 ◦ C.
Polyclonal rabbit and mouse anti-Crimean-Congo hemorrhagic
fever virus sera were generated in rabbits and mice immunized
with inactivated Crimean-Congo hemorrhagic fever virus TurkeyKelkit06 strain.
The entire protocol was approved by the local ethics committee
of the Firat University.
2.2. Human sera
Serum samples from 12 patients, including one fatality, were
obtained from the Refik Saydam National Public Health Agency,
The National Crimean-Congo Hemorrhagic Fever Reference Center
of Turkey. All patients had laboratory-confirmed Crimean-Congo
hemorrhagic fever infection and acquired the virus during the 2007
outbreaks in Turkey’s Kelkit Valley region.
27
washes in TBST, goat anti-mouse ␤-gal conjugate, diluted 1:1500
in TBST (Southern Biotech, USA), was added to each well, and
the plate was incubated for 1 h at room temperature with gentle rocking. After extensive washing, the substrates NBT (nitro
blue tetrazolium) and X-gal (5-bromo-4-choloro-3-indolyl-betad-galactopyranoside) were added to each well and incubated at
37 ◦ C. To avoid overdevelopment, microplates were checked every
10 min under a microscope. When infected cells appeared medium
blue to dark purple in color, TBST was added to stop the reaction. All
handling of virus was performed in a biosafety level 3 laboratory.
2.4. Focus formation assay
Crimean-Congo hemorrhagic fever Turkey-Kelkit06 stock
viruses were titrated by the focus formation assay following the
peroxidase–antiperoxidase method described by Okuna et al. with
minor modifications (Okuno et al., 1985a, 1985b). Vero E6 or SW13 cells were seeded into 96-well plates (Corning, USA) and grown
to 90% confluency. For the Crimean-Congo hemorrhagic fever virus
Turkey-Kelkit06 infectivity assay, a microplate with triplicate wells
containing 10-fold diluted virus stock was incubated at 37 ◦ C with
5% CO2 for 1 h. The viral inocula were removed and washed with
PBS, and the cell monolayer was covered with virus medium containing 1% CMC (Sigma–Aldrich, Germany) then incubated at 37 ◦ C
and 5% CO2 for 24 h. The medium was removed, and the cells were
washed with PBS, fixed with 10% neutral buffered formaldehyde
at room temperature for 20 min, and washed twice with TBST. The
cells were permeated with 1% Nonidet-P 40 (NP-40) in TBST for
30 min with gentle rocking, and blocked with 5% skim milk in TBST.
Infected cells were probed with rabbit anti-Crimean-Congo hemorrhagic fever virus polyclonal antisera (1:1500) and incubated for
1 h at room temperature with gentle rocking. The cells were treated
with goat anti-rabbit IgG (1:1000; Sigma–Aldrich, Germany) and
rabbit peroxidase–antiperoxidase (PAP) (1:200; Sigma–Aldrich,
Germany). Each step was preceded by washing the wells 3–4 times
with TBST. Finally, viral antigens in infected cells were detected
by staining with 3,3n -diaminobenzidine tetrahydrochloride (DAB)
(Sigma–Aldrich, Germany) and H2 O2 for 10–20 min at room temperature. The DAB solution was removed, and the cells were rinsed
with deionized water to stop the reaction. The stained cells (the
foci) were examined microscopically.
2.5. Fluorescent immunofocus assay
2.3. Pseudo-plaque assay
The pseudo-plaque assay was based on the FastPlaxTM Titer
Kit (Novagen, USA) used in the baculovirus expression system.
Crimean-Congo hemorrhagic fever virus Turkey-Kelkit06 stock
viruses were titrated by the pseudo-plaque assay with some modifications (Mitchell et al., 2010). Vero E6 or SW-13 cells were
grown to confluence in 96-well microtiter plates (Corning, USA)
in DMEM containing 10% FBS at 37 ◦ C with 5% CO2 for 18–24 h.
The cells were washed twice with 10 mM phosphate buffer saline
(PBS, pH 7.2) and incubated with 10-fold serially diluted virus
suspension in triplicate wells at 37 ◦ C with 5% CO2 for 1 h. After
incubation, the inoculum was removed and the cell monolayer
was overlaid with virus medium containing 1% carboxymethyl
cellulose (CMC) (Sigma–Aldrich, Germany) incubated at 37 ◦ C
with 5% CO2 for 8–24 h. The cells were fixed with 10% neutral buffered formaldehyde for 20 min, washed twice with TBST
(100 mM Tris–HCl pH 8.0, 1.5 M NaCl, 1% Tween 20), permeabilized with 0.1% Triton X 100 in PBS for 20 min with gentle rocking
and then blocked with 5% skim milk in PBS. Polyclonal mouse antiCrimean-Congo hemorrhagic fever virus serum diluted 1:1500 in
TBST was added to each well, and the plate was incubated for 1 h
at room temperature with gentle rocking. Following three 10-min
Lab Tek II 8-well chamber slides (Sigma–Aldrich, Germany)
were seeded with Vero E6 or SW-13 cells at a density of 3 × 105
and incubated at 37 ◦ C with 5% CO2 to produce a confluent monolayer. The cell monolayers were inoculated with 10-fold serially
diluted virus. After absorption for 1 h at 37 ◦ C, the supernatants
were removed and the cells were washed with PBS. The cell
monolayer was overlaid with virus medium containing 1% CMC
then incubated 37 ◦ C with 5% CO2 for 8 to 24 h. After fixation
with 10% neutral buffered formaldehyde at room temperature for
20 min, the cells were permeated with 0.1% Triton X 100 in PBS
for 20 min with gentle rocking and blocked with 5% skim milk in
PBS. Slides were moved to a moist chamber, incubated with rabbit
anti-Crimean-Congo hemorrhagic fever virus polyclonal antisera
(1:1000) for 1 h in TBST at 37 ◦ C and washed 3 times with TBST.
Antibody-labeled cells were detected after cells were incubated
for 1 h with goat anti-rabbit IgG conjugated with fluorescein isothiocyanate (FITCH) (Southern Biotech, USA) and diluted 1:1000
in TBST and then washed three times with TBST and once with
distiled water. The cells were mounted in anti-fading medium
(Sigma–Aldrich, Germany) and analyzed by immunofluorescence
microscopy (Olympus BX50, Japan). Images were captured with
Olympus DP72 digital camera and Olympus DP2-BSW software.
28
E. Berber et al. / Journal of Virological Methods 187 (2013) 26–31
Fluorescent foci that were located within the 0.7 cm2 area of each
well were counted, and virus titers were calculated and expressed
as fluorescent focus units per ml.
new cells with succeeding generations. Smaller satellite foci, probably resulting from dissemination of progeny virus from the initial
site of infection, surrounded the primary plaques (Fig. 2A–D).
2.6. Statistical analysis
3.2. Time course for the development of pseudo-plaque assay
Statistical analyses were conducted using Graph Pad Prism, v
4.0.
A dilution of Crimean-Congo hemorrhagic fever virus TurkeyKelkit06 known to produce 30–50 pseudo-plaques per well was
infected, fixed and stained 2–5 days PI. There were no detectable
pseudo-plaques on PI day 2 (Fig. 3). Pseudo-plaques were visible
3–5 days PI and increased each day in size but not in number (Fig. 3).
Even though the pseudo-plaques were not visible until 3 days PI by
naked eye, we were able to detect the pseudo-plaques at 24 h PI
under ordinary light microscopy.
3. Results
3.1. Titration of Crimean-Congo hemorrhagic fever virus
Turkey-Kelkit06 in VERO E6 and SW-13 cells by pseudo-plaque
assay
Vero E6 and SW-13 cells were grown to confluence in 24well cell culture plates and infected with 10-fold serial dilutions
of virus. Cells were fixed 5 days PI, and blue pseudo-plaques
were detected with polyclonal mouse anti-Crimean-Congo hemorrhagic fever virus serum following degradation of X-gal by
␤-galactosidase linked to secondary antibodies. The pseudo-plaque
titers of Crimean-Congo hemorrhagic fever virus Turkey-Kelkit06
in Vero E6 were not significantly different from viral titers in SW13 cells; mean Crimean-Congo hemorrhagic fever virus titers were
slightly greater in SW-13 cells (5.2 versus 5.1 log10 PPA/ml). In the
SW-13 cell line, the pseudo-plaques were less distinct than in the
Vero E6 cell line, but they were still quantifiable (Fig. 1A and B).
The intensity of staining was strongest at the center of each
pseudo-plaque and gradually decreased with increased distance
from the center, indicating that the virus had spread and infected
3.3. Linear relationship between dilution and virus titer in the
pseudo-plaque assay
After optimizing pseudo-plaque assay on 24-well plates for
quantitation of Crimean-Congo hemorrhagic fever virus TurkeyKelkit06, Vero E6 or SW-13 cells were seeded into 96-well tissue
culture plates. The cell monolayers were incubated with 10 fold
serially diluted virus and overlaid with virus medium containing 1%
CMC. Cells were fixed at 24 h PI and processed for pseudo-plaque
assay. The pseudo-plaques were counted under light microscope.
The pseudo-plaque titers of Crimean-Congo hemorrhagic fever
virus Turkey-Kelkit06 in Vero E6 and SW-13 cells were 4.3 × 105
and 5.1 × 105 PPA/ml, respectively. These data indicated that 96well plates were suitable for determining the titer of the virus by
pseudo-plaque assay.
To determine a correlation between dilution and virus titer by
pseudo-plaque assay, Vero E6 and SW-13 cells were infected with
10-fold dilutions of the virus, and the pseudo-plaques were counted
under the microscope 24 h PI by pseudo-plaque assay. Fig. 4 shows
the linear relationship (log 10) between virus dilutions and the
number of pseudo-plaques per well. The number of pseudo-plaques
counted for each Crimean-Congo hemorrhagic fever virus dilution
in this assay showed low variation among replicates.
3.4. Comparison of virus titer as determined by the
pseudo-plaque assay, fluorescent immunofocus assays, and focus
formation assay
Pseudo-plaque assay accuracy was measured by comparing
pseudo-plaque assay titers with the titers of the focus formation
assay and the fluorescent immunofocus assay. For this purpose,
Vero E6 and SW-13 cells were seeded into 96-well plates for the
pseudo-plaque assay and the focus formation assay and into 8well chamber slides for the fluorescent immunofocus assay. Serial
10-fold dilutions of three different stocks of Crimean-Congo hemorrhagic fever virus Turkey-Kelkit06 were inoculated onto Vero E6
and SW-13 cells and overlaid with virus medium containing 1%
CMC. Cells were fixed at 24 h PI and processed as described (Fig. 5).
The three stocks exhibited approximate virus titers of 104 and 105
infectious units (Table 1). The Kruskal–Wallis test showed no significant differences among the assays (P > 0.05).
3.5. Application of the pseudo-plaque assay for clinical
investigation
Fig. 1. Dose-dependent response for pseudo-plaque titration of Crimean-Congo
hemorrhagic fever virus Turkey-Kelkit06. Vero E6 (A) and SW-13 (B) cells were
infected with 10-fold dilutions of Crimean-Congo hemorrhagic fever virus TurkeyKelkit06 in duplicate, fixed 5 days PI, and then stained for Crimean-Congo
hemorrhagic fever virus Turkey-Kelkit06. Viral titers were determined by counting
pseudo-plaques at 10−4 dilutions.
Crimean-Congo hemorrhagic fever confirmed human sera from
12 individuals was investigated by pseudo-plaque assay and fluorescent immunofocus assay. Vero E6 cells were incubated with
50 ␮l of 5-fold diluted sera in triplicate wells and overlaid with
the medium containing 1% CMC. Cells were fixed 8 or 24 h PI and
processed for pseudo-plaque assay and fluorescent immunofocus
E. Berber et al. / Journal of Virological Methods 187 (2013) 26–31
29
Fig. 2. Prototypical morphologies of Crimean-Congo hemorrhagic fever virus Turkey-Kelkit06 pseudo-plaques. Vero E6 (A and B) and SW-13 (C and D) cells were infected
with Crimean-Congo hemorrhagic fever virus Turkey-Kelkit06 and then fixed 24 h PI (A and C; magnification: 100×) and 5 days PI (B and D; magnification: 40×). The presence
of polyclonal mouse anti-Crimean-Congo hemorrhagic fever virus serum-stained pseudo-plaques was determined using light microscopy.
assay (Fig. 6). Six of the 12 samples were found to be positive at
8 h PI by pseudo-plaque assay, and 10 were found to be positive
at 24 h PI. At 8 h PI, there were no positive samples by fluorescent
immunofocus assay, while seven samples were positive at 24 h PI.
4. Discussion
Accurate titration of viruses is a crucial step in the field of virology. To date, several methods have been used for viral titration
depending on the nature of the virus (Counihan et al., 2006; Cruz
and Shin, 2007; Payne et al., 2006; Shepherd et al., 1986; Yang
et al., 1998). Plaque forming unit (PFU) assays and endpoint titration assays are the most common. Both PFU and endpoint titration
assays for a 50% tissue culture infectious dose (TCID50) require a
monolayer of cells susceptible to virus infection and that develop
a virus-induced cytopathic effect (CPE) (Dulbecco and Vogt, 1954;
Reed and Muench, 1938). These traditional methods cannot be used
to quantify viruses that produce little or no CPE.
Crimean-Congo hemorrhagic fever virus often tends to develop
a noncytopathic persistent infection of cell cultures, depending on
the strain of the virus. Formation of plaques and CPE may take place
after serial passage of the virus (Shepherd et al., 1986). If the virus
Fig. 3. Course of development of visible Crimean-Congo hemorrhagic fever virus Turkey-Kelkit06 pseudo-plaques. Vero E6 cells infected with Crimean-Congo hemorrhagic
fever virus Turkey-Kelkit06 were fixed and stained at the indicated times post-infection.
30
E. Berber et al. / Journal of Virological Methods 187 (2013) 26–31
Fig. 4. Linear relationship between virus dilutions and number of pseudo-plaques per well. Vero E6 cells (A) and SW-13 cells (B) in 96-well microplates were infected with
serially diluted Crimean-Congo hemorrhagic fever virus Turkey-Kelkit06 and stained by pseudo-plaque assay 24 h PI; correlation coefficient, r = −0.8814 (A), and −0.8879
(B).
Fig. 5. Detection of Crimean-Congo hemorrhagic fever virus Turkey-Kelkit06 using pseudo-plaque assay, focus formation assay and fluorescent immunofocus assay. CrimeanCongo hemorrhagic fever virus Turkey-Kelkit06-infected Vero E6 cells were fixed at 24 h and stained with polyclonal mouse anti-Crimean-Congo hemorrhagic fever virus
serum and secondary antibody conjugated with ␤-gal (A); stained with rabbit anti-Crimean-Congo hemorrhagic fever virus serum and PAP complex (B); or stained with
polyclonal mouse anti-Crimean-Congo hemorrhagic fever virus serum and secondary antibody conjugated with FITCH (C).
Fig. 6. Comparison of pseudo-plaque and fluorescent immunofocus assays with a Crimean-Congo hemorrhagic fever confirmed human serum. Crimean-Congo hemorrhagic
fever infection was detected 8 h PI and 24 h PI by pseudo-plaque assay (A). Fluorescent immunofocus assay detected Crimean-Congo hemorrhagic fever infection only 24 h
PI (B) (NC; mock infected Vero E6 cells 24 h PI).
Table 1
Titration with focus formation assay (FFA), fluorescent immunofocus assay (FIFA), and pseudo-plaque assay (PPA) on Vero E6 and SW-13 cells.
Virusa
Vero E6 cells
b
Stock 1
Stock 2
Stock 3
a
b
FFA
3.8 × 105
3.4 × 104
2.5 × 105
SW-13 cells
FIFA
PPA
FFA
FIFA
PPA
2.1 × 105
2.0 × 104
3.4 × 105
4.5 × 105
3.8 × 104
3.3 × 105
3.5 × 105
4.0 × 104
2.0 × 105
2.2 × 105
8.0 × 103
2.3 × 105
5.5 × 105
6.3 × 104
2.2 × 105
Stocks of Crimean-Congo hemorrhagic fever virus Turkey-Kelkit06.
Viral titer (log10 FFA, FIFA, or PPA/ml).
E. Berber et al. / Journal of Virological Methods 187 (2013) 26–31
does not develop plaques, virus titration must be conducted by
immunofluorescence assay with specific polyclonal or monoclonal
antibodies.
A pseudo-plaque assay for titration and clonal isolation of
adeno-associated viruses was described by Mitchell et al. (2010).
Pseudo-plaque assay relies on the NBT and X-gal staining method,
which enables visualization of infected cells by probing the cells
with specific antbodies. Enzyme-catalyzed color development of
infected cells is detected, not directly from plaque formation, but
from antibody-mediated detection of viral antigens. A major advantage of pseudo-plaque assay is that, since the method relies on
detection of viral antigens in infected cells using virus specificantibodies, it does not require the development of CPE in the
infected cell monolayer. The pseudo-plaque assay in 96-well
microtiter plates to titrate Crimean-Congo hemorrhagic fever virus
Turkey-Kelkit06 in Vero E6 and SW-13 cells proved to be a simple
and rapid means of analyzing a large number of samples. Results
were obtained 24 h after cell infection, a reduction in titration times
from 3–5 days.
The plaque assay is the gold standard for quantitation of
Crimean-Congo hemorrhagic fever virus. Researchers have used
fluorescent immunofocus assay to determine viral infectivity
of Crimean-Congo hemorrhagic fever virus. These studies were
performed with a liquid overlay and, after 24 h, cell infection fluorescent foci were used to calculate titration (Connolly-Andersen
et al., 2007, 2009), possibly allowing a secondary infection, which
could affect titration results. In this pseudo-plaque assay, an
overlay of CMC was used to prevent a secondary infection.
The pseudo-plaque assay accuracy was confirmed by comparing its results with focus formation and fluorescent immunofocus
titers using 3 stocks of the virus. No significant differences
were observed in the virus titers across the three methods
(Table 1).
No difference in sensitivity was observed in virus detection
in the cell-adapted strain of Crimean-Congo hemorrhagic fever
virus Kelkit06, but pseudo-plaque assay was more sensitive than
the fluorescent immunofocus assay in detecting the virus from
primary isolates of Crimean-Congo hemorrhagic fever virus from
humans.
The pseudo-plaque assay was shown to be suitable for titration
of Crimean-Congo hemorrhagic fever virus Kelkit06, which does
not develop plaques, suggesting that it might also be used for other
viruses. With minor modifications, the method could also be used
for applications such as serotyping of viruses and measurement of
virus-specific neutralizing antibodies.
Acknowledgments
This research was financially supported by and approved by the
Scientific and Technological Research Council of Turkey (TUBITAK),
project number 108G126.
References
Burt, F.J., Leman, P.A., Smith, J.F., Swanepoel, R., 1998. The use of a reverse
transcription-polymerase chain reaction for the detection of viral nucleic acid
in the diagnosis of Crimean-Congo haemorrhagic fever. Journal of Virological
Methods 70, 129–137.
31
Connolly-Andersen, A.M., Magnusson, K.E., Mirazimi, A., 2007. Baso-lateral entry and
release of Crimean-Congo hemorrhagic fever virus in polarized MDCK-1 cells.
Journal of Virology 81, 2158–2164.
Connolly-Andersen, A.M., Douagi, I., Kraus, A.A., Mirazimi, A., 2009. Crimean-Congo
hemorrhagic fever virus infects human monocyte-derived dendritic cells. Virology 390, 157–162.
Counihan, N.A., Daniel, L.M., Chojnacki, J., Anderson, D.A., 2006. Infrared fluorescent immunofocus assay (IR-FIFA) for the quantitation of noncytopathic and
minimally cytopathic viruses. Journal of Virological Methods 133, 62–69.
Cruz, D.J.M., Shin, H., 2007. Application of a focus formation assay for detection and
titration of porcine epidemic diarrhea virus. Journal of Virological Methods 145,
56–61.
Donets, M.A., Rezapkin, G.V., Ivanov, A.P., Tkachenko, E.A., 1982. Immunosorbent
assays for diagnosis of Crimean-Congo hemorrhagic fever (CCHF). American
Journal of Tropical Medicine and Hygiene 31, 156–162.
Dulbecco, R., Vogt, M., 1954. Plaque formation and isolation of pure lines with
poliomyelitis viruses. Journal of Experimental Medicine 99, 167–182.
Ergonul, O., 2006. Crimean-Congo haemorrhagic fever. Lancet Infectious Diseases 6,
203–214.
Hoogstraal, H., 1979. The epidemiology of tick-borne Crimean-Congo hemorrhagic
fever in Asia, Europe, and Africa. Journal of Medical Entomology 15, 307–417
(review).
Karti, S.S., Odabasi, Z., Korten, V., Yilmaz, M., Sonmez, M., Caylan, R., Akdogan, E.,
Eren, N., Koksal, I., Ovali, E., 2004. Crimean-Congo hemorrhagic fever in Turkey.
Emerging Infectious Diseases 10, 1379–1384.
Leibovitz, A., McCombs 3rd., W.M., Johnston, D., McCoy, C.E., Stinson, J.C., 1973. New
human cancer cell culture lines. I. SW-13, small-cell carcinoma of the adrenal
cortex. Journal of the National Cancer Institute 51, 691–697.
Maltezou, H.C., Andonova, L., Andraghetti, R., Bouloy, M., Ergonul, O., Jongejan, F.,
Klatches, N., Nichol, S., Niedrig, M., Platonov, A., Thomson, G., Leitmeyer, K.,
Zeller, H., 2010. Crimean-Congo hemorrhagic fever in Europe: current situation
calls for preparedness. Eurosurveillance 15, 19504.
Mitchell, D.A.J., Lerch, T.F., Hare, J.T., Chapman, M.S., 2010. A pseudo-plaque method
for infectious particle assay and clonal isolation of adeno-associated virus. Journal of Virological Methods 170, 9–15.
Okuno, Y., Yamanishi, K., Lwin, S., Takahashi, M., 1985a. Micro-neutralization
test for mumps virus using the 96-well tissue culture plate and PAP
(peroxidase–antiperoxidase) staining technique. Microbiology and Immunology 29, 327–335.
Okuno, Y., Fukunaga, T., Tadano, M., Okamoto, Y., Ohnishi, T., Takagi, M., 1985b. Rapid
focus reduction neutralization test of Japanese encephalitis virus in microtiter
system. Archives of Virology 86, 129–135.
Ozdarendeli, A., Aydin, K., Tonbak, S., Aktas, M., Altay, K., Koksal, I., Bolat, Y., Dumanli,
N., Kalkan, A., 2008. Genetic analysis of the M RNA segment of Crimean-Congo
hemorrhagic fever virus strains in Turkey. Archives of Virology 153, 37–44.
Ozdarendeli, A., Canako˘glu, N., Berber, E., Aydin, K., Tonbak, S., Ertek, M., Buzgan, T.,
Bolat, Y., Aktas¸, M., Kalkan, A., 2010. The complete genome analysis of CrimeanCongo hemorrhagic fever virus isolated in Turkey. Virus Research 147, 288–293.
Papa, A., Bino, S., Llagami, A., Brahimaj, B., Papadimitriou, E., Pavlidou, V., Velo, E.,
Cahani, G., Hajdini, M., Pilaca, A., 2002. Crimean-Congo hemorrhagic fever in
Albania. European Journal of Clinical Microbiology and Infectious Diseases 21,
603–606.
Payne, A.F., Binduga-Gajewska, I., Kauffman, E.B., Kramer, L.D., 2006. Quantitation
of flaviviruses by fluorescent focus assay. Journal of Virological Methods 134,
183–189.
Reed, L.J., Muench, H., 1938. A simple method of estimating fifty percent endpoints.
American Journal of Hygiene 27, 493–497.
Saijo, M., Tang, Q., Niikura, M., Maeda, A., Ikegami, T., Prehaud, C., Kurane,
I., Morikawa, S., 2002b. An immunofluorescence technique for detection of
immunoglobulin G antibodies to Crimean-Congo hemorrhagic fever virus using
HeLa cells expressing recombinant nucleoprotein. Journal of Clinical Microbiology 40, 372–375.
Shepherd, A.J., Swanepoel, R., Leman, P.A., Shepherd, S.P., 1986. Comparison of methods for isolation and titration of Crimean-Congo hemorrhagic fever virus. Journal
of Clinical Microbiology 24, 654–656.
Tonbak, S., Aktas, M., Altay, K., Azkur, Aydin, K., Kalkan, A., Bolat, Y., Dumanli, N.,
Ozdarendeli, A., 2006. Crimean-Congo hemorrhagic fever virus: genetic analysis
and tick survey in Turkey. Journal of Clinical Virology 44, 4124–4420.
Whitehouse, C.A., 2004. Crimean-Congo hemorrhagic fever. Antiviral Research 64,
145–160.
Yang, D.P., Goldberg, K.M., Xiang-Dong, M., Magargle, W., Rapparort, R., 1998.
Development of a fluorescent focus identification assay using serotype-specific
monoclonal antibodies for detection and quantitation of rotaviruses in a
tetravalent rotavirus vaccine. Clinical and Diagnostic Laboratory Immunology
5, 780–783.