Detection of Infectious Rotaviruses by Flow Cytometry Albert Bosch, Rosa M. Pintó, Jaume Comas, and Francesc-Xavier Abad

1. Introduction

Human rotaviruses are considered the main cause of viral gastroenteritis in infants and young children throughout the world (1). Their transmission is through the fecal-oral route, mostly after ingestion of contaminated water and food (2). Since an extremely high number of virus particles are present in the feces during the acute gastroenteritis, methods based on electron microscopy, passive particle agglutination tests, or enzyme-linked immunosorbent assays are readily employed for clinical diagnosis. However, the sensitivity of these procedures is not high enough to detect the low number of viral particles sometimes present in the environment (3). In the case of environmental samples, amplification of viral nucleic acids by polymerase chain reaction assays coupled to reverse transcription (RT-PCR) has been increasingly applied to detect rotaviruses in water (4) and shellfish samples (5). However, procedures based on molecular approaches have to face the drawback that they do not differentiate between infectious and noninfectious particles, which is of major relevance from the public health point of view.

Virus propagation in cell culture prior to detection by immunological or molecular procedures accomplishes the dual purpose of increasing the amount of target material and incorporating an infectivity assay as well.

Wild-type rotaviruses present difficulties in their in vitro replication, although some of them may be adapted to grow in several cell lines such as the monkey kidney cell line MA104 or the human intestinal cell line CaCo-2 (6,7). More than a decade ago, an assay for the specific detection of infectious rotaviruses in environmental samples, involving an indirect immunofluorescence test (IIF) and optical microscopy (OM) counting of infected foci in infected MA-104 cell monolayers, was described (4). On the other hand, CaCo-2 cells have been successfully employed in our laboratory for infectivity assays of several fastidious enteric virus strains present in water samples (8).

Flow cytometry (FCM) is a method to quantify components or study the structural characteristics of cells, mainly by optical means. The term flow cytometry derives from the measurement of single cells as they flow in a fluid stream through a measuring point surrounded by several detectors. FCM involves the use of a beam of light projected through a liquid stream that contains cells. As the cells cross the focused light, they emit signals that are captured by detectors. These signals are then converted for computer storage and data analysis and can provide information about cellular properties. These biophysical properties are then correlated with biological and biochemical properties of interest. To make the measurement of biological properties of concern, the cells are usually stained with fluorescent dyes that specifically bind to cellular constituents. Sorting instruments may also isolate specific cells according to their cytometric profile. FCM can be thus employed for counting and specific cell sorting with several cell types.

The use of FCM for the detection of virally infected cells presents several advantages with respect to OM detection and has been used for different viruses (9-11). The IIF-OM detection method, although efficient, is cumbersome and requires well-trained personnel, whereas FCM is an automatable procedure that allows the processing of a large number of samples. Additionally, most major hospitals and public health institutions in developed countries possess a flow cytometer. However, standardization of an FCM method requires the study of several critical points, such as fixation (type of fixative, contact time, and so on) or minimization of background noise (lowering the antibody working solution concentration, assaying different blocking solutions, and so on).

2. Materials

2.1. Infectivity Assay

1. Horizontal laminar flow hood.

2. Vertical laminar flow hood.

3. CO2 incubator.

4. CaCo-2 cell line, a human colon adenocarcinoma cell line, used at passage level 80-100.

5. A cytopathogenic strain of human rotavirus, i.e., Wa or Itor P13, used as positive control.

6. Eagle's minimum essential medium (MEM) with Earle's salts; Auto-Pow® (ICN, Costa Mesa, CA, cat. no. 1110024) as the cell culture medium. Dissolve and sterilize according to the manufacturer's instructions, and supplement with the following reagents at final concentrations: 0.15% NaHCO3, 15 mM HEPES, 2 mM L-glutamine, 100 U/mL penicillin, 100 ^g/mL streptomycin.

7. Fetal calf serum (FCS; BioWhittaker, Walkerville, MD).

8. Phosphate buffer solution (PBS): dissolve 8 g NaCl, 0.2 g KCl, 1.15 g Na2HPO4, and 0.2 g KH2PO4 in 1000 mL deionized water. After complete dissolution, adjust the pH to 7.1-7.2 by 1 M HCl addition. Sterilize by autoclaving and store at 4 ± 1°C.

9. Trypsin for cell passage: dissolve 2.5 g of trypsin (tissue culture grade 1:250; DIFCO, Sparks, MD, cat. no. 0152-13) and 0.2 g of EDTA in 1000 mL PBS. After complete dissolution sterilize by filtration through 0.22-^m GS-type filters (Millipore, Bedford, MA; cat. no. GSWP047S0) and store at -20°C until use.

10. Trypsin for rotavirus activation: dissolve 0.1 g of trypsin grade IX (Sigma, St. Louis, MO; cat. no. T-0134) in 10 mL PBS. Sterilize by filtration. Prepare a stock solution of 1 mg/mL, by subsequent 1:10 dilution with PBS. Store in 1-mL aliquots at -20°C.

11. Sterile plastic ware: 5- and 10-mL plastic pipets, 58 x 17-mm Petri dishes (Nunc, Roskilde, Denmark; cat. no. 150288), sterile 0.2- and 1.0-mL tips, and 10-mL plastic tubes.

2.2. Indirect Immunofluorescence

1. Orbital vertical mixer (Selecta, Barcelona, Spain) or similar.

2. 1 M Sucrose solution in deionized water. Sterilize by filtration through 0.22-^m filters and store at room temperature.

3. ^-Formaldehyde solution (20%): dissolve 20 g^-formaldehyde (Merck, cat. no. 1.04005.) in 70 mL deionized water. Heat the mixture for 1 h at 80°C in a thermostatic bath inside an extraction cabinet. Add 2-3 drops of 1 M NaOH. After 1 h more of heating, filter the mixture through Whatman filter paper. Bring to a final volume of 100 mL with deionized water and store at -20°C.

4. 1 M Dipotassium phosphate solution in deionized water: filter through a 0.45-^m filter and autoclave. Store at room temperature.

5. 1 M Sodium phosphate solution in deionized water: filter through a 0.45-^m filter and sterilize by autoclaving. Store at room temperature.

6. 1 M Phosphate buffer: adjust the pH of 80 mL 1 M dipotassium phosphate solution to pH 7.4 with 1 M sodium phosphate solution.

7. 0.2 M Phosphate buffer: add 20 mL of 1 M phosphate buffer to 80 mL of sterile deionized water.

8. Saline solution: add 0.8 g of NaCl in 100 mL deionized water. Sterilize by autoclaving and store at 4 ± 1°C.

9. Fixative: mix, at room temperature, 3 mL of 20% ^-formaldehyde solution, 1.2 mL of 1 M sucrose, 10 mL of 0.2 M phosphate buffer, and 5.8 mL of deionized water.

10. Blocking solution: the day of IIF assay, dissolve 5 g powdered skim milk and 0.2 mL of a 10% Triton X-100 solution in 100 mL of saline solution.

11. Rotavirus positive control serum (Institute Virion, Ruschlikon, Switzerland; cat. no. 3193) as primary antibody: Reconstitute the lyophilized powder with 0.4 mL of deionized water. Transfer the volume to a plastic tube and add blocking solution to reach the working dilution recommended by the manufacturer (between 1:32 and 1:110). Prepare the same day of experiment.

12. Fluorescein isothiocyanate (FITC)-labeled rabbit anti-human IgG (Sigma; cat. no. F-4512) as secondary antibody: Prepare a 1:400 working dilution with blocking solution. Prepare the same day of experiment.

2.3. FCM Detection

1. Coulter Epics XL flow cytometer (Beckman-Coulter, Miami, FL) or similar.

3. Methods

3.1. Infectivity Assay

1. Prepare CaCo-2 cell monolayers in a laminar flow hood by trypsinization at a split ratio of 1:3. Cells are used when they are confluent or near confluence. In normal growing conditions, a maximum of 20 58 x 17-mm Petri dishes may be produced from a 175-cm2

culture flask after trypsinization (see Subheading 2.1., item 9). The cells are grown in cell culture medium with 10% (v/v) FCS.

2. Samples (see Note 1) are pretreated with 10 ^g/mL trypsin (see Note 2) for 30 min at 37°C .

3. Wash the cells monolayers twice, using 5 mL of cell culture medium per Petri dish to remove all traces of FCS (see Note 3).

4. Remove the washing medium and add 200 ^L of each trypsin-treated sample per Petri dish. As positive controls inoculate 200 ^L of a viral suspension of a cytopathogenic rotavirus strain with an original titer of 103-104 infectious units per mL in each of two Petri dishes. As negative controls inoculate 200 ^L serum-free medium over each of four Petri dishes.

5. Incubate for 60 min at 37°C, with two or three gentle swirlings of the inoculum over the monolayer.

6. Add 5 mL per plate of postinfection overlay medium consisting of serum-free cell culture medium supplemented with 5 ^g/mL trypsin (see Note 4).

7. Incubate Petri dishes for 4 d at 37°C in an atmosphere of 5% CO2-air.

3.2. Indirect Immunofluorescence

1. Recover the cells by vigorously pipeting up and down the postinfection medium, and transfer the whole volume to a 10-mL plastic tub.

2. Centrifuge at low speed, around 900g for 10 min.

3. Carefully discard the supernatant.

4. Resuspend cell pellet in 1 mL of saline.

5. Transfer the cell suspension to a 1.5-mL microtube.

6. Centrifuge microtubes in an Eppendorf 5415C microcentrifuge (or similar) at 10,000g for 90 s, at room temperature (see Note 5).

7. Discard the supernatant by aspiration using a micropipet and resuspend again the cell pellet with another 1 mL of saline.

8. Spin the microtube again at the same conditions described above.

9. Discard the supernatant and resuspend cell pellet with 1 mL of freshly prepared (the same day) fixative solution.

10. Incubate in gentle agitation for 30 min at room temperature.

11. Centrifuge at 10,000g for 90 s, at room temperature. Carefully discard the fixative and add 1 mL of saline to each microtube (see Note 6).

12. Perform two more washes with saline as described above (1 mL of saline solution per microtube, centrifuge at previous described conditions, discard the supernatant, add 1 mL more of saline solution, centrifuge another time, and pipet out the supernatant).

13. Permeabilize the fixed cells by a 15-min treatment with 0.1% Triton X-100 at room temperature, with constant gentle agitation.

14. Add 300 ^L of primary antibody solution to the cell pellet of each microtube, with gentle mixing (see Note 7). Place the microtubes in an orbital vertical mixer.

15. Keep in agitation for 45 min at room temperature (see Note 8).

16. Centrifuge the microtubes at 10,000g for 90 s, and discard the primary antibody solution by aspiration with a pipet.

17. Wash the cellular pellet five times with the blocking solution, using for each wash 1 mL of blocking solution per microtube (see Note 7), centrifuge after each wash at 10,000g for 90 s, and gently discard the supernatant by careful aspiration with a pipet.

18. Add 300 ^L of the FITC-labeled secondary antibody solution. Place the microtubes in an orbital vertical mixer.

19. Keep in agitation for 45 min in the dark at room temperature (see Note 8).

20. Centrifuge at 10,000g for 90 s, and discard the secondary antibody solution by careful aspiration with a pipette.

21. Wash the cell pellet at least four times with saline solution, using for each wash 1 mL per microtube (see Note 7), centrifuge after each wash at 10,000g for 90 s, and gently discard the supernatant by careful aspiration with a pipet.

22. Resuspend the cell pellet in 1 mL of saline.

23. Store at 4 ± 1°C in dark conditions to await the FCM assay (see Note 9).

3.2. FCM Detection

The cellular suspensions are analyzed with a Coulter Epics XL flow cytometer equipped with the standard 488-nm argon-ion laser at 15 mW power.

1. Select logarithmic forward angle light scatter (FSC), logarithmic side-angle light scatter (SS), logarithmic green fluorescence (FL1), and time as the acquisition parameters.

2. Adjust cytometer settings according to the values given in Table 1.

3. Define an FS log vs SS log dotplot (resolution: 128 x 128 channels) (Fig. 1A) and a 1024-channel histogram using the FL1 parameter (Fig. 1B).

4. Check instrument stability, analyzing 10-^m calibration beads (Flowcheck, Coulter). Record the channel position of these beads for FSC, SSC, and FL1 as a daily quality control of the instrument.

5. Acquire a minimum of 100,000 cells. To avoid coincidences, injection flow rate must be adjusted to keep total events/s below 800.

6. Draw a polygonal region on the cellular population of the FSC vs SS dotplot. FSC is used to select cell size, and SS is used to select shape and structure in order to restrain the readings to the population of intact eukaryotic cells and not the cell debris.

7. Create a gate using the polygonal region of the FSC/SS dotplot. Apply the gate on the fluorescence histogram (Fig. 1 B) in order to represent fluorescence of the cells inside this region. Fluorescence intensity (x-axis) is expressed in log scale, which means that in the higher channels small differences in channel number represent large differences in the amount of dye per cell.

8. Define a cursor on the FL1 histogram from 59.7 to 1024. This cursor is used to quantify positive events. The XL analysis software give us the percentage of cells under the cursor. The position of this cursor is established after analysis of a pool of negative control samples (11). For quality assurance, a large number (30-50 n) of mock-infected samples should be processed before attempting the detection of wild-type rotaviruses in natural environmental samples. An arbitrary cursor (A) is drawn at the right end of their fluorescence curves (channels 10-1024). This cursor included some of the negative cell population counts. The mean fluorescence of each of the A cursors from the negative controls is calculated. These mean values follow a normal distribution. The mean and standard deviation of this latter curve are figured, and a second cursor (B) is then defined starting at the point obtained by adding 2 standard deviations to the mean fluorescence (channel 60), and ending at channel 1024. The ratio of cells present in cursor B in respect to the total counted cells is calculated for each negative sample, and the mean plus 2 standard deviations of these ratios in the negative samples is established as the threshold of positivity .

Table 1

XL Cytometer Settings




Total gain

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