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Appl Environ Microbiol, July 1998, p. 2392-2396, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Flow Cytometry Detection of Infectious Rotaviruses
in Environmental and Clinical Samples
F. Xavier
Abad,
Rosa M.
Pintó, and
Albert
Bosch*
Department of Microbiology, University of
Barcelona, 08028 Barcelona, Spain
Received 24 June 1997/Accepted 31 March 1998
 |
ABSTRACT |
A method for the detection of infectious human rotaviruses based on
infection of CaCo-2 cells and detection of infected cells by indirect
immunofluorescence and flow cytometry (IIF-FC) has been developed. The
technique was validated by performing a seminested reverse
transcription-PCR assay with sorted cell populations. The efficiency of
the procedure has been compared with that of the standard method of
infection of MA104 cells and ulterior detection by IIF and optical
microscopy (IIF-OM) and with that of infection of MA104 cells and
detection by IIF-FC. The limit of sensitivity for the detection of the
cell-adapted strain Itor P13, expressed as the most
probable number of cytopathogenic units, was established as 200 and 2 for MA104 and CaCo-2 cells, respectively, by the IIF-FC method. The
ratio of infectious virus particles to total virus particles for a
wild-type rotavirus was determined to be 1/2 × 106
and 1/2 × 104 for IIF-OM with MA104 cells and IIF-FC
with CaCo-2 cells, respectively. The use of IIF-FC with CaCo-2 cells
was tested with fecal and water samples and proved to be more effective
than the standard procedure for rotavirus detection.
| |
INTRODUCTION |
Human rotaviruses, the main cause of
viral gastroenteritis in children, are transmitted by the fecal-oral
route and may be acquired through ingestion of contaminated water and
food (4). The most widely used techniques for the diagnosis
of rotaviral diarrhea include electron microscopy (3),
immunological methods such as latex agglutination or enzyme
immunosorbent assays (5), and molecular techniques
(6). However, these methods do not differentiate between
infectious and noninfectious particles, and this difference is
important from the point of view of public health, vaccine production,
and antiviral research. The inclusion of an infectivity test prior to
the specific detection solves not only this problem but also that of
the lack of the sensitivity that is required for some kind of samples,
such as environmental samples. Wild-type rotaviruses present
difficulties in regard to their in vitro replication, although some of
them may be adapted to grow in several cell lines, such as the human
intestinal cell line CaCo-2 or the monkey kidney cell line MA104
(9). The standard methods for the diagnosis of specific
infectious rotaviruses involve immunofluorescence tests and optical
microscopy (OM) counting of infected foci in the culture
(9). In the present work an alternative method for the
quantification of infectious rotaviruses, based on the use of flow
cytometry (FC), is described. The technique was applied to detect
wild-type rotaviruses from fecal or water samples.
| |
MATERIALS AND METHODS |
Cell cultures and viral strains.
MA104 cells, derived from
fetal monkey kidney, and CaCo-2 cells, derived from a human colon
carcinoma, were propagated in Eagle's minimal essential medium
supplemented with fetal calf serum (FCS) at concentrations of 5 and
10%, respectively. The cytopathic strain Itor P13
(13) of group A human rotavirus, type 3, was used throughout these studies for the standardization of the technique.
Fecal samples.
Wild-type rotaviruses were obtained from
stool samples from 2- to 12-month-old children with acute diarrhea from
the Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, and
identified by latex agglutination (Rotalex; Orion Diagnostica, Espoo,
Finland). All but one of these samples contained rotaviruses that were
noncultivable in MA104 cells. Viruses were chloroform extracted from
0.1 g of stool sample and suspended in 1 ml of phosphate-buffered
saline. The number of extracted viral particles was determined for some samples by the colloidal gold and negative-staining transmission electron microscopy method (8).
Water samples.
Wild-type rotaviruses were also isolated from
water samples supplied by drinking water companies from different
cities throughout Spain. Viruses were concentrated on location from 500 liters of river water by filtration through positively charged Zeta
Plus MK II filters (AMF Cunó, Sefiltra, Alcobendas, Spain),
elution with 500 ml of 3% beef extract-0.05 M glycine buffer, and
organic flocculation to a final volume of 50 ml by the procedures
described elsewhere (2). Rotavirus diagnosis was performed
by infecting MA104 cells with water concentrates and detecting the
infected cells by an indirect immunofluorescence (IIF) test.
Viral infections.
Wild-type rotavirus infections were
performed with MA104 and CaCo-2 cells (11). Virus inocula
were pretreated with trypsin (Difco, Detroit, Mich.; tissue culture
grade 1:250) at a concentration of 10 µg/ml for 30 min at 37°C, and
after adsorption a serum-free overlay medium containing 5 µg of
trypsin per ml was added. Infections proceeded for 2 to 5 days, and
cells were processed for IIF as described below. Viral enumerations of
the cell-adapted strain Itor P13 were performed as
previously described (11) and expressed as the most probable
number of cytopathogenic units (MPNCU) per milliliter. All experiments
involving detection and titration were performed at least in
triplicate.
IIF-OM detection in MA104 cells.
The IIF-OM has been
previously described (2) and has been applied to detect
infectious rotavirus in water. Briefly, it consisted of infections of
monolayers of cells grown in microtiter plates with 20 µl of inoculum
per well, fixation of infected cells with cold (4°C) 80% acetone,
incubation with human rotavirus antiserum (Institute Virion Ltd.,
Ruschlikon, Switzerland) diluted 1/100, and then staining with goat
anti-human immunoglobulins conjugated to fluorescein isothiocyanate
(Sigma-Aldrich, Alcobendas, Spain) diluted 1/32. As a blocking reagent,
2% FCS was used. The plates were examined inverted with a Nikon
microscope fitted for epifluorescence. A total volume of 1.6 ml of each
sample was assayed.
IIF-FC detection in MA104 and CaCo-2 cells.
The
Itor P13 strain of human rotavirus was used to develop the
IIF-FC detection procedure. In this procedure, cells grown in 60-mm-diameter dishes were inoculated with 200 µl of the sample. After the infection period, cells were mechanically detached, washed,
and treated with different fixatives, such as cold (4°C) 80% acetone
for 30 min, cold (
20°C) 70% ethanol for 30 min, cold (4°C)
acetone-ethanol (4:6) for 30 min, and cold (4°C)
acetone-methanol-formalin (1:1:1) for 1 min. An IIF assay was performed
as described above but with the secondary antibody diluted 1/400. As
blocking reagents, both 5% nonfat milk and 2% FCS were tested. The
cellular suspensions were analyzed in a Coulter Epics Elite flow
cytometer equipped with a 488-nm argon-ion laser at 15-mW power and
with a combination of 550 DL and 525 BP filters in order to recover the
green fluorescence of fluorescein. The forward-angle light scatter was
used to select cell size, and the side-angle light scatter was used to
select shape and structure, so as to restrict the readings to the
population of intact eukaryotic cells and not the cell debris. Data
obtained were represented in diagrams that displayed the distribution
of the population of cells among 1,024 channels of fluorescence
intensity. The height (y axis) indicated the number of cell
counts with a particular level of fluorescence. Fluorescence intensity
(x axis) was expressed on a log scale, which means that
small differences in channel number represent large differences in the
amount of dye per cell. The total number of cells counted was around
500,000. For quality assurance, 40 mock-infected samples were processed for IIF-FC, and an arbitrary cursor (A) was drawn at the right end of
their fluorescence curves (channels 10 to 1024). This cursor included
2% of the negative cell population counts. The mean fluorescence of
each of the A cursors from the 40 negative controls was calculated. These mean values followed a normal distribution. The mean and standard
deviation of this curve were calculated, and a second cursor (B) was
then defined, starting at the point obtained by adding two standard
deviations to the mean fluorescence (channel 60) and ending at channel
1024. The ratio of cells present in cursor B to total counted cells was
calculated for each negative sample, and the mean plus two standard
deviations of these ratios in the 40 negative samples was established
as the threshold of positivity (0.00112 and 0.00061 for MA104 and
CaCo-2 cells, respectively). The procedure was validated by using
10-fold dilutions of the Itor P13 strain to infect
monolayers of MA104 and CaCo-2 cells. Different infectious doses were
assayed, and each of them was determined at least in triplicate for
each cell type. When wild-type viruses were assayed, negative and
positive control samples were included in each assay.
Nucleic acid detection in sorted cell populations by a seminested
RT-PCR technique.
Nucleic acid detection was performed with sorted
cell populations by a seminested reverse transcription-PCR (RT-PCR)
technique. Infected CaCo-2 cells were processed by FC as described
above and sorted by combining their scatter and fluorescence
characteristics. The fluid stream was broken into droplets that were
electrically charged and deflected into a collection vessel by passage
through an electric field. Three different populations were sorted:
8,000 cells from the population included in cursor A, 800 cells from the population included in cursor B, and 80,000 cells from the rest of
the population. A two-step RT-PCR was then performed by standard
procedures as described elsewhere (7). Briefly, primers Beg9
and End9 were used for an initial RT-PCR to obtain the full-length gene
9 segment (1,062 bp). Primer RVG9 and primer aET3 were used to obtain,
by a subsequent seminested PCR amplification, the characteristic 374-bp
fragment of human rotavirus type 3. RT of RNA was carried out in
20-µl reaction mixtures. Ten microliters of eluates containing RNA
extracted from the samples was heated at 99°C for 5 min and immediately placed on ice, and 10 µl of 2× RT reaction mix was added
and incubated for 1 h at 43°C. The RT reaction mix consisted of
50 mM Tris hydrochloride (pH 8.3), 40 mM KCl, 7 mM MgCl2, 1 mM each primer (Beg9 and End9), 200 mM deoxynucleoside triphosphates (dNTPs), and 2 U of avian myeloblastosis virus reverse transcriptase. Forty microliters of PCR mix was added to 10 µl of the RT product, to
yield a 50-µl mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl,
1.5 mM MgCl2, 1 mM each primer (Beg9 and End9), 200 mM dNTPs, and 2 U of Taq polymerase. The mixture was initially
denatured at 94°C for 4 min and then subjected to 40 cycles of
amplification, each consisting of 1 min at 94°C, 2 min at 47°C, and
5 min at 72°C. A final extension was carried out at 72°C for 10 min. Ten microliters of the amplified product was used as the template in the seminested PCR. Primer and dNTP concentrations were 0.5 and 100 mM, respectively. Thirty cycles of amplification were performed under
the same conditions as before but with an increase in the annealing
temperature, to 50°C. Twenty microliters of each sample was analyzed
in an agarose gel in order to observe the expected 374-bp band of the
PCR product.
| |
RESULTS |
IIF-FC detection of the Itor P13 strain.
For the
development of the IIF-FC detection method for rotavirus-infected
cells, several fixatives were tested in order to permeabilize the cells
to the different antibodies. Among all the fixatives,
acetone-methanol-formalin (1:1:1) provided the best specific
signal/background ratio. Two kinds of blocking reagents were assayed,
and 5% nonfat milk was found to be the one of choice, since a better
blocking activity in terms of background levels and more consistent
results from experiment to experiment were achieved with nonfat milk
than with FCS.
Tenfold dilutions of viral stocks of the Itor P13 strain
containing 4 × 107 MPNCU/ml were assayed by IIF-FC
after infection of monolayers of MA104 and CaCo-2 cells. CaCo-2 cells
offered enhanced sensitivity in comparison with MA104 cells, as the
minimum amounts of infectious rotavirus detected were 2 and 200 MPNCU,
respectively (Fig. 1). The mean ratios,
from three separate experiments, of the number of cells in cursor B
(Fig. 1) to the total number of counted cells after infection with 200 MPNCU of virus were 0.003750 ± 0.004770 and 0.023123 ± 0.015138 for MA104 and CaCo-2 cells, respectively. This means that
after infection at a multiplicity of infection of 0.0001, 0.375 and
2.3% of the MA104 and CaCo-2 cell populations, respectively, became
positive.
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FIG. 1.
Rotavirus detection by IIF-FC. Detection of
Itor P13 virus in MA104 cells (left column) and CaCo-2
cells (right column) is shown. The inoculum used (MPNCU) is indicated
in the upper right corner of each plot. Cursors A and B are defined in
the text. Plots represent a typical data set.
|
|
Validation of the IIF-FC rotavirus detection method with CaCo-2
cells.
To confirm the validity of the IIF-FC method for the
detection of rotavirus-infected CaCo-2 cells, viral RNA was detected by
means of an RT-PCR method in sorted cell populations. After infection
with 200 MPNCU of the Itor P13 strain, all three sorted
populations (cells from cursor A, cells from cursor B, and cells from
the rest of the population) contained rotavirus RNA (Fig.
2), while after infection with 2 MPNCU,
only the cells included in cursors A and B were positive (Fig. 2). None
of the three populations was positive for the presence of rotavirus RNA
after infection with 0.2 MPNCU (Fig. 2).
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FIG. 2.
Rotavirus RNA detection by a seminested RT-PCR after
infection of CaCo-2 cells with different inocula of the
Itor P13 strain and cell sorting. Lanes: 1, mock-infected
cells from cursor A; 2, mock-infected cells from cursor B; 3, mock-infected cells from the rest of the population; 4, cells from
cursor A after infection with 0.2 MPNCU; 5, cells from cursor B after
infection with 0.2 MPNCU; 6, cells from the rest of the population
after infection with 0.2 MPNCU; 7, cells from cursor A after infection
with 2 MPNCU; 8, cells from cursor B after infection with 2 MPNCU; 9, cells from the rest of the population after infection with 2 MPNCU; 10, cells from cursor A after infection with 200 MPNCU; 11, cells from
cursor B after infection with 200 MPNCU; 12, cells from the rest of the
population after infection with 200 MPNCU; M, molecular size marker;
, negative control RT-PCR; +, positive control. Cursors A and B are
defined in the text. Numbers on the left and right indicate base
pairs.
|
|
IIF-FC detection of rotaviruses in feces.
Eight fecal samples
containing noncultivable rotaviruses and one fecal sample with
rotaviruses growing in MA104 cells were assayed by IIF-FC. Two of the
eight noncultivable samples became positive by IIF-FC after infection
of CaCo-2 cells (Table 1). The sample
with cultivable rotaviruses was also positive by this procedure. The
number of physical particles present in this sample (812F) was
evaluated and found to be 108 particles per g of extracted
feces. The limit of sensitivity of IIF-FC for this sample was at a
stool suspension dilution of 10
2, which means that the
ratio of infectious particles to physical particles is 1/2 × 104. The same sample (812F) evaluated by IIF-OM detection
with MA104 cells showed 50 fluorescent foci per g of extracted feces,
which means a ratio of infectious particles to physical particles of 1/2 × 106. The characteristic profiles for positive
and negative stool samples are shown in Fig.
3.
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FIG. 3.
Detection of wild-type rotavirus in CaCo-2 cells. The
sample (Table 1) is indicated in the upper right corner of each plot.
Cursors A and B are defined in the text.
|
|
IIF-FC detection of waterborne rotaviruses.
Twenty-four water
sample concentrates were analyzed by IIF-FC after infection of CaCo-2
cells, and the results were compared with the data provided by IIF-OM
detection after infection of MA104 cells (Table 1 and Fig. 3). Ten and
six of these samples were positive by IIF-FC and IIF-OM, respectively.
Five samples were positive by both methods. The one discordant sample
became positive by the IIF-FC method with CaCo-2 cells after
ultracentrifugation of a 1.6-ml sample (the volume used in the MA104
system), resuspension in 200 µl, and cell inoculation of the latter
amount. The characteristic profiles for positive and negative water
samples are shown in Fig. 3.
| |
DISCUSSION |
The clinical diagnosis of rotaviral gastroenteritis can be
performed on the basis of detection of physical particles. However, the
determination of infectious viruses is of critical importance in some
situations, such as the monitoring of water for the prevention of
waterborne outbreaks of rotaviral gastroenteritis (1, 2). Other situations where control of the infectivity of samples is also
required include vaccine production and the development of antiviral
and disinfectant agents. The poor replicating capacity of rotaviruses
in cell culture has prompted the use of cell-adapted strains in most
studies involving antiviral (10, 15) and disinfection (14) research. The method described here could be of
relevant application in these areas and enables the use of cell
culture-adapted strains and some wild-type strains as well. The
development of this method relied on the use of the CaCo-2 cell line
and ulterior FC for the detection of infected cells. CaCo-2 cells
showed higher sensitivity to rotavirus infection than MA104 cells,
either with the cell-adapted strain Itor P13 or with
wild-type viruses. The sensitivity of CaCo-2 cells was 2 log units
higher than that of MA104 cells. With the wild-type strains the higher
sensitivity was determined from the higher number of positive stool and
water samples detected and from the higher ratio of infectious
particles to total virus particles observed in the 812 stool samples.
The higher sensitivity of CaCo-2 cells to rotavirus infections has also
been previously described (9, 11). The sensitivity of IIF-FC
with CaCo-2 cells could be increased by concentrating the sample prior
to cell inoculation. The use of FC for the detection of virus-infected
cells has several advantages over OM detection and has been used for
different viruses (12, 16). The IIF-OM detection method,
although efficient, is cumbersome and requires well-trained personnel,
while IIF-FC is an automatable procedure. Most major hospitals and
public health institutions in developed countries own a flow cytometer.
The standardization of the IIF-FC methodology requires the study of several critical points, such as fixation and minimization of the
background noise. As a fixative, the best choice in our assay was the
mixture of acetone, methanol, and formalin, and as a blocking reagent,
the solution of 5% nonfat milk was the most effective in reducing
background fluorescence. The antibody concentration also played an
important role in lowering the background, and the working dilutions
were adjusted in the IIF-FC procedure with respect to those in the
IIF-OM detection method in order to achieve a good ratio of specific
labeling to background. The method described here has been found to be
highly useful and reproducible for the specific detection of infectious
wild-type rotaviruses and enables the processing of a large number of
samples. Highly sensitive detection procedures are also required for
the detection of very low numbers of rotaviruses in some environmental
situations in order to accurately assess the level of risk posed by
their presence.
| |
ACKNOWLEDGMENTS |
We acknowledge the skillful assistance of J. Comas of the
Flow Cytometry Unit of the Scientific and Technical Services of the
University of Barcelona. We are grateful to N. Margall, Servei de
Microbiologia, Hospital de la Santa Creu i Sant Pau, for providing stool samples.
This work was supported in part by grant 1995SGR00197 from the CIRIT,
Generalitat de Catalunya. F. X. Abad has a PQS contract from the
Generalitat de Catalunya.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dep.
Microbiology, University of Barcelona, School of Biology, Av. Diagonal,
645, 08028 Barcelona, Spain. Phone: (34 93) 402.14.85. Fax: (34 93)
411.05.92. E-mail: albert{at}bio.ub.es.
| |
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Appl Environ Microbiol, July 1998, p. 2392-2396, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
