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Concentrating rotaviruses from water samples using monolithic chromatographic supports
Journal of Chromatography A, 1216 (2009) 2700–2704
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Concentrating rotaviruses from water samples using monolithic chromatographic supports Ion Gutiérrez-Aguirre a,∗ , Marko Banjac b , Andrej Steyer c , Mateja Poljˇsak-Prijatelj c , b ˇ Matjaˇz Peterka b , Aleˇs Strancar , Maja Ravnikar a a b c
Department of Biotechnology and Systems Biology, National Institute of Biology, Veˇcna pot 111, SI-1000 Ljubljana, Slovenia BIA Separations, d.o.o., Teslova 30, SI-1000 Ljubljana, Slovenia Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Zaloˇska 4, SI-1000 Ljubljana, Slovenia
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Article history: Available online 6 November 2008 Keywords: Rotavirus Methacrylate monoliths Concentrating viruses Ion-exchange chromatography Water contamination RT q-PCR
a b s t r a c t Rotaviruses are the leading cause of diarrhoea in infants around the globe and, under certain conditions they can be present in drinking water sources and systems. Ingestion of 10–100 viral particles is enough to cause disease, emphasizing the need for sensitive diagnostic methods. In this study we have optimized the concentration of rotavirus particles using methacrylate monolithic chromatographic supports. Different surface chemistries and mobile phases were tested. A strong anion exchanger and phosphate buffer (pH 7) resulted in the highest recoveries after elution of the bound virus with 1 M NaCl. Using this approach, rotavirus particles spiked in 1 l volumes of tap or river water were efficiently concentrated. The developed concentration method in combination with a real time quantitative polymerase chain reaction assay detected rotavirus concentrations as low as 100 rotavirus particles/ml. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Rotaviruses are the main aetiological agent of acute viral gastroenteritis in young children around the globe [1]. They are responsible for 500 000–600 000 deaths each year, which primarily affect developing countries [2]. The infectious dose for rotaviruses, as for other enteric viruses, is extremely low, and as little as 10 viral particles can originate a gastroenteritis episode [3,4]. Rotaviruses are introduced in aquatic environments (river, lake, sea. . .), most commonly through leaking sewer systems, leaking septic systems, urban runoff and agricultural runoff [5]. Moreover rotavirus can contaminate drinking water supplies under defined conditions, such as, earthquakes and floods or intentional release. In such cases, undetectable but still infectious circulating concentrations of rotavirus constitute a risk to human health. Classic rotavirus detection methods consist of enzyme linked immunosorbent assays (ELISA), electron microscopy (EM) or polymerase chain reaction (PCR) [6,7]. Recently, a reverse transcription quantitative PCR (RT-qPCR) assay was developed, which offered improved sensitivity over other methods, detecting as low as 1 × 104 rotavirus particles/ml in river and tap water samples [8]. Still, for the detection of the low amounts of viruses expected
∗ Corresponding author. Tel.: +386 1 4233388; fax: +386 1 2573847. E-mail address: [email protected] (I. Gutiérrez-Aguirre). 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.10.106
in water samples, an initial concentration step has to be utilized. Methods used for concentrating viruses from water samples include: different filtration procedures based on the size of the virus (tangential flow filtration, vortex flow filtration) and/or adsorption–elution methods based on the surface charge of the virus (glass wool, positively or negatively charged filters) [9–13]. The concentrated fraction obtained with such methods has to be applied to a secondary concentration step which typically involves ultracentrifugation, further ultrafiltration or flocculation of the viruses. In this way 1–1000 l samples can be concentrated to 1–10 ml volumes and utilized for molecular or alternative detection techniques. Different concentration methods with their advantages and disadvantages are reviewed by Fong and Lipp [5]. The problems that arise when concentrating viruses from water samples usually depend on the method and include, high cost, unsuitability for large volumes, need for a secondary concentration step, clogging (filters), low recoveries, inhibitors transmitted to posterior molecular detection steps, etc. [5]. Because most enteric viruses are negatively charged at ambient pH, adsorption–elution with an electropositive filter is one of the most commonly used techniques [12], however electropositive filters are easily clogged and under defined conditions, such as marine water samples, they result on low recoveries [14]. In this work we used methacrylate monoliths to bind and concentrate rotaviruses from water samples. Methacrylate mono-
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liths, commercialized by BIA Separations under the name CIM (convective interaction media), are made of a single block of highly porous polymer, where the mass transfer, unlike traditional beadbased chromatography, is based on convective flow, allowing a more rapid and efficient exchange between mobile and stationary phases [15]. Furthermore, the large diameter (up to 1500 nm) of the highly interconnected channels integrating the monoliths favours the circulation and interaction with large biomolecules such as viruses [15]. The methacrylate monoliths can bear different ligands on the surface allowing their use for anionic/cationic exchange, affinity, and hydrophobic interaction chromatography [15]. CIM monoliths have already been successfully applied for the binding, separation and/or concentration of different viruses, such as bacteriophage T4 [16], influenza [17], measles and mumps [18] and tomato mosaic virus (ToMV) [19,20]. In order to optimize the binding of rotaviruses to CIM, different anion-exchange (QA) and cation-exchange (SO3 ) chemistries, as well as different pH and elution conditions were tested using disk format (bed volume 0.34 ml). The chromatographic fractions were characterized by EM and ELISA, and the viral concentration in each fraction was quantified by RT-qPCR. The optimized conditions were used in concentration experiments using artificially inoculated 1 l tap or river water samples applied to CIM tube format (bed volume 8 ml).
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hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffer pH 7, 50 mM phosphate buffer pH 7, 50 mM acetate buffer pH 4 and 50 mM citrate buffer pH 9. Different NaCl concentrations used in linear gradient as well as stepwise elutions are indicated in the figures and tables. The loaded samples were always prepared from rotavirus positive clarified 10% stool suspensions of known concentration estimated by counting under EM [21]. In order to dilute the traces of NaCl inherent to the clarified stool, which could interfere with the virus binding to the ion-exchange support, the initial sample was always diluted at least 10-fold in the mobile phase (buffer A) before loading to the CIM disk. All load, flow through and elution fractions were characterized with EM and ELISA, and rotavirus amount was quantified with RT-qPCR as described below. To calculate the capacity of the QA monolith to bind rotavirus, a stool suspension of known rotavirus concentration was continuously loaded into a CIM QA disk at 6.2 ml/min. Flow through was collected in 10 ml fractions, and fractions were applied to rotavirus specific ELISA detection. The first ELISA positive fraction was considered as 0% breakthrough, and the capacity of rotavirus particles/ml of monolithic support was calculated accordingly. CIM monolithic columns were regenerated and stored as recommended by the manufacturer. 2.3. Quantification of rotavirus in chromatographic fractions by RT-qPCR
2. Experimental 2.1. Characterization of rotavirus positive stool samples and fractions: EM and ELISA Rotavirus positive stool samples were obtained from children hospitalized with acute gastroenteritis. Samples were characterized, clarified and 10% stool suspensions prepared as described by Gutiérrez-Aguirre et al. [8]. The concentration of rotavirus in the initial 10% stool suspension was estimated by virus counting under an electron microscope JEM 1200 EXII (Jeol, Tokyo, Japan) using the latex-negative staining technique [21]. Such samples were used for preparing the loads used in the optimizations of the rotavirus binding to CIM, and for inoculating the tap and river water samples used in concentration experiments. EM was also used to evaluate the integrity of the viruses eluted from the CIM monolithic columns. A rotavirus specific ELISA assay (Premier Rotaclone, Meridian Bioscience, Cincinnati, OH, USA) was used for fast monitoring of chromatographic fractions for rotavirus presence. 2.2. Optimization of the rotavirus binding to CIM Strong anion- and cation-exchange CIM disk monolithic columns (BIA Separations, Ljubljana, Slovenia) with a bed volume of 0.34 ml, a diameter of 12 mm and a length of 3 mm, were used to optimize the virus binding to the support as well as the posterior elution. As anion exchanger the positively charged quaternary amine (QA) surface chemistry was chosen, while as cation exchanger negatively charged sulfuric anhydride (SO3 ) was utilized. The disks were placed in a specially designed housing (BIA Separations, Ljubljana, Slovenia) and connected to a gradient HPLC system (Knauer, Berlin, Germany). The system was composed of two K-500 pumps, a UV–vis detector K-2500 operating at 280 nm with a 10 mm optical path and 10 l volume flow cell connected by 0.25 mm i.d. polyether ether ketone (PEEK) capillary tubes. An in-line conductivity monitor (Amersham Biosciences, Uppsala, Sweden) was used to monitor the ionic strength of the mobile phase. HPLC software from Knauer was used in all experiments. Unless otherwise indicated, flow rate was set to 2 ml/min. Different mobile phases were tested: 50 mM (4-(2-
For the quantification of the rotavirus amount present in each chromatographic fraction a recently developed RT-qPCR was used [8]. In all experiments RNA from the known-concentration stool suspension (used for load preparation), was isolated and 10-fold serially diluted from neat to 1 × 10−6 . Each dilution was then applied to rotavirus specific RT-qPCR in triplicate, and threshold cycles (PCR cycle in which the real time amplification curve becomes exponential = Ct) were calculated, in order to build a standard curve of log(concentration) vs Ct [8]. In addition, the RNA from the fractions generated after each chromatographic run (load—L, flow through—FT, elution 1—E1, elution 2—E2) was also isolated and applied in triplicate to rotavirus specific RT-qPCR. The Ct obtained for each fraction was used to estimate the rotavirus concentration via the previously generated standard curve. For the concentration experiments in tap and river water samples, known concentration (2 ng) of luciferase RNA was added to the samples as an external control immediately prior to the RNA isolation. Each RNA dilution was then applied not only to rotavirus specific RT-qPCR but also to luciferase specific RT-qPCR [8]. The Ct obtained for luciferase were used to normalize the rotavirus specific Ct, to account for variations due to other factors (rather than solely the virus concentration), such as, differences in RNA extraction yield or presence of PCR inhibitors. In all experiments RNA was isolated from 250 l of sample using TRIzol reagent, according to manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA) 2.4. Concentration experiments A rotavirus positive stool suspension of known concentration was used as seed to inoculate samples consisting of 1 l of tap or river water to different final concentrations. These samples were pumped through a CIM QA 8 ml tube column using a Milton Roy LMI B71 dosing pump (Milton Roy Europe, Point Saint Pierre, France) operating at 100 ml/min flow rate. At least 10 column volumes (CVs) of 50 mM phosphate pH 7 were used to wash any unbound compound from the CIM QA tube after the loading, and then the bound viruses were eluted by using 1 M NaCl in the same buffer. Elution fraction volume was always between 10 and 15 ml. The elution was monitored spectrophotometrically at 254 nm with a Smartline
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preparative UV Detector 200 (Knauer) equipped with a preparative flow cell. All connections were made using HPLC grade PEEK tubing of appropriate i.d. for preparative applications and corresponding fittings. In the case of river water samples a 0.45 m cut off–142 mm i.d. filter (Sartorius, Goettingen, Germany) in a specially designed metallic housing, was placed between the pump and the CIM QA tube. Samples before and after concentration were applied to RT-qPCR detection and quantification as described above, and concentration factors were calculated. For detection purposes, samples giving a positive Ct value in the rotavirus specific RT-qPCR in at least two of the three replicates were considered as rotavirus positive. Eight ml CIM tube monolithic columns were regenerated and stored as recommended by the manufacturer. 3. Results 3.1. Binding of rotavirus to CIM monolithic columns First, we tested the binding of the virus at neutral pH (pH 7) using a strong anion exchanger (QA) and a strong cation exchanger (SO3 ). Four ml of a stool sample (1.64 × 1010 particles/ml of G2P [4] rotavirus) 10-fold diluted in buffer A (HEPES 50 mM pH 7) were loaded on both disk QA and SO3 monolithic columns, and eluted with a gradient of 0–2 M NaCl in buffer A (Fig. 1). In the case of the QA disk, rotaviruses were detected by ELISA only in the elution peak (E1), and not in the flow through (FT), while the opposite occurred when using SO3 chemistry (Fig. 1). Therefore one can conclude that rotaviruses bind efficiently to CIM QA monoliths, as it was expected taking into account that most enteric viruses are negatively charged at ambient pH [12]. From the gradient shown in the upper panel of Fig. 1, it seems that most of the bound material is eluted at NaCl concentrations between 0.1 and 0.3 M NaCl. In order to obtain a direct proof of the integrity of the eluted virus, FT and E1 fractions were characterized by EM, and the intact rotavirus particles were observed indicating no change in their shape and structure as they pass through the monolithic support (Fig. 1, lower panel). Next, we decided to determine the capacity of the QA monolithic support to bind rotaviruses present in a real stool sample. A rotavirus stool of 2.66 × 1010 particles/ml was continuously loaded into a CIM QA disk and the flow through was collected in 10 ml fractions, which were applied to rotavirus detection by ELISA. The first rotavirus positive fraction was considered as the 0% breakthrough of the capacity, and this occurred in the sixth fraction indicating that we were able to load 50 ml of the stool without going beyond capacity. This resulted in a capacity estimation of 4 × 1012 rotavirus particles/ml of CIM QA monolith for a real stool suspension at 0% breakthrough. 3.2. Optimization of rotavirus recovery from QA monoliths Prior to concentration experiments using higher water volumes, conditions, which enable the highest viral recoveries needed to be determined, i.e., mobile phase buffer composition, pH and NaCl concentration in elution buffer. Different runs with different conditions were performed (Table 1). In all cases the runs consisted of a FT followed by two step gradient elutions (Fig. 2). The first elution peak (E1) was obtained by eluting with 0.6, 0.8 or 1 M NaCl concentration in buffer A, while the second elution peak (E2) was always obtained by eluting with 2 M NaCl (in order to release the material that remained attached to CIM after first elution, see Table 1). The amount of rotavirus present in the loaded sample as well as in FT, E1 and E2 fractions was quantified using RT-qPCR, and percentages of viral recovery were calculated (Table 2). In all the tested runs the amount of rotavirus in the FT fractions was 0% (Table 2) indicating that under all conditions CIM QA is able to efficiently
Fig. 1. Chromatographic runs on disk CIM QA and CIM SO3 monolithic columns. (Upper panel) A stool containing rotavirus G2P [4], as estimated by EM counting, at 1.64 × 1010 particles/ml was diluted 10-fold in buffer A (HEPES 50 mM, pH 7) and 4 ml loaded into a disk CIM QA monolithic column. Flow rate was kept at 2 ml/min. After a wash step using buffer A, a linear gradient 0–100% of buffer B (HEPES 50 mM, NaCl 2 M, pH 7) was applied. Flow through (FT) and elution peaks (E1) are indicated in the panel. Both FT and E1 were applied to rotavirus specific ELISA detection and results (+ or −) are indicated in the panel. The thick line represents A280 , while the thin line represents the conductivity. (Middle panel) Same as in upper panel except the disk monolithic column used, which in this case was SO3 . (Lower panel) Electron microscope micrograph of one of the fractions corresponding to the E1 peak in the upper panel.
bind the viruses. The viral recoveries obtained in the first elution peak varied depending on the NaCl concentration used, namely 40%, 86%, and 91% of the loaded virus was recovered when eluting with 0.6, 0.8 and 1 M NaCl, respectively (Tables 1 and 2, runs 1–3). In runs 4 and 5, we evaluated the effect on the recovery of using basic and acid pH in the mobile phase. The use of sodium acetate buffer pH 4 and sodium citrate buffer pH 9 resulted in a 30% and 15% decrease, respectively, of the recovery in comparison to HEPES buffer pH 7 (Tables 1 and 2, runs 3–5). In the last run
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Table 1 Run conditions tested in disk CIM QA columns. Conditions Run
Loada
Flow rate
Buffer A
pH
[NaCl] Bb
[NaCl] C2
1 2 3 4 5 6
4 ml 4 ml 4 ml 4 ml 4 ml 10 ml
2 ml/min 2 ml/min 2 ml/min 2 ml/min 2 ml/min 6 ml/min
HEPES 50 mM HEPES 50 mM HEPES 50 mM Acetate 50 mM Citrate 50 mM Phosphate 50 mM
7 7 7 4 9 7
0.6 M 0.8 M 1M 1M 1M 1M
2M 2M 2M 2M 2M 2M
a Load was in all cases prepared by 10× diluting in the corresponding buffer A, a stool suspension containing rotavirus G2P [4] at 1.64 × 1010 particles/ml. b Concentration of NaCl included in buffer A in order to get buffer B (first elution step) and buffer C (second elution step).
Table 2 Rotavirus recoveries from the chromatographic runs listed in Table 1. Run
1 2 3 4 5 6
Fig. 2. Typical rotavirus binding and step gradient elution using disk CIM QA monolithic column. Run conditions and virus recoveries are those shown for run number 6 in Tables 1 and 2.
Virus recovery (%) FT
E1
E2
Total
0 0 0 0 0 0
40.7 86.8 91.1 58 76 99.8
13.3 1.48 0.8 3.8 0.7 2.2
54 88.3 92 61.8 76.7 102
(run 6) the buffer was changed to phosphate pH 7, and the load and flow rate were increased to 10 ml and 6 ml/min, respectively. These changes improved recovery of the virus at values close to 100% in the first elution peak (Tables 1 and 2, run 6) (Fig. 2). Therefore, in following experiments with higher volume samples we used run 6 conditions: phosphate buffer 50 mM, pH 7 and an elution step using 1 M NaCl. 3.3. Concentration of rotavirus from spiked tap and river water samples To determine the ability of CIM QA monoliths to concentrate low numbers of rotaviruses present in tap or river waters, we inoculated 1 l samples of the mentioned waters to different rotavirus concentrations. The HPLC system and the CIM QA disk monolithic columns were substituted by a dosage membrane pump operating at 100 ml/min, a modular preparative UV detector and CIM QA 8 ml tube monolithic columns. A 0.45 m pore size filter was placed between the pump and the column in the cases when river water was tested to prevent larger particles from getting into the col-
umn. The pH of the water samples was adjusted by adding 500 mM phosphate buffer, pH 7, in a 1:10 (v/v) ratio. All the load and elution fractions were applied to RT-qPCR detection and quantification and where applicable, concentration factors were estimated (Table 3). The volume of the elution peak was in all the cases close to 15 ml meaning that if starting from a 1000 ml sample, with a 100% effective concentration, a concentration factor of 65 times would be expected. Out of the three tap water samples tested before the concentration step, rotaviruses were detected in two (9.7 × 106 and 9.7 × 103 particles/ml). RT-qPCR was unable to detect viruses in the tap water of the third sample seeded to a final concentration of 97 particles/ml; this was only possible after the concentration using CIM tube. For the first two samples we were able to calculate concentration factors, which ranged from 56 to 66× (Table 3), indicating a very efficient viral recovery, as it was already observed when working with CIM QA disk format. Concerning river water samples, RT-qPCR could detect rotavirus down to 9.7 × 102 particles/ml, coinciding with the detection limit of the method, Ct ≈ 37 [8] (Table 3). After concentration using CIM QA tube we were able to confidently detect both 9.7 × 102 and 97 particles/ml. The calculated concentration factors ranged from 20 to 40× (Table 3). The decrease in the concentration yield in comparison with tap water is most probably due to the presence of the in-line filter which in contrast to tap water experiments generates a surface where a portion of the viruses can unspecifically adsorb. Still, the procedure was enough efficient to enable the detection of samples, which were otherwise beyond the limit of detection of RT-qPCR.
Table 3 Concentration of rotaviruses spiked in 1-l samples of tap and river water. Spike [RoV] (particles/ml)
Ct load
Ct concentrate
Concentration factora
Tap water
9.7 × 10 9.7 × 103 9.7 × 101 Non-spiked
23.9 (+) 34.1 (+) Non-detected Non-detected
19.1 (+) 28.4 (+) 33.6 (+) Non-detected
66× 56× nmb nm
River water
9.7 × 106 9.7 × 105 9.7 × 104 9.7 × 103 9.7 × 102 9.7 × 101 Non-spiked
24.5 (+) 27.8 (+) 32 (+) 34.6 (+) 37.7 (+) Non-detected Non-detected
19.2 (+) 19.6 (+) 23.5 (+) 26.7 (+) 35.8 (+) 36.6 (+) Non-detected
22× 40× 40× 20× nm nm nm
6
a b
Concentration factor was calculated using RT-qPCR for quantifying the virus concentration both the load and the concentrate as indicated in Section 2. Non-measurable. Concentration factor for samples giving Ct values higher than 35 (beyond limit of quantification) was not measured.
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4. Discussion The objective of our study was to use CIM chromatographic supports for fast and efficient concentration of rotaviruses that, under specific circumstances, are present in tap and environmental water samples. Classic concentration methods consist mainly of the use of ultrafiltration units, glass wool, polyethyleneglycol (PEG) precipitation, ultracentrifugation, flocculation and binding to charged membranes [9–13]. Lately, the use of charged membranes is becoming one of the most utilized methods for concentration of viruses in water samples [5]. Adsorption–elution to electropositive membranes is, for example, the designated method for concentration of enteric viruses from drinking waters by the Information Collection Rule of the US Environmental Protection Agency (http://www.epa.gov/nerlcwww/icrmicro.pdf). The relative simplicity of their use and the ability to process large water volumes are among the advantages of such membranes [5]. Low recoveries in some cases, use of elution buffers with components that can inhibit posterior PCR detection (high pH + beef extract) and need of an additional secondary concentration are among the disadvantages [5]. In this work we took advantage of the properties of the CIM monolithic supports and apply them to the concentration of rotaviruses from different water samples. The highly porous (macropores and mesopores) structure of the CIM monolithic support provides a high surface area accessible to the viral particles resulting in very high binding capacities and recoveries [15]. We obtained a very high binding capacity of 4 × 1012 rotavirus particles/ml of CIM QA monolith for a clarified stool suspension at 0% breakthrough, therefore, as much as 3.2 × 1013 particles of rotaviruses could be accommodated into a CIM 8 ml QA tube. Such high viral binding capacities make CIM monoliths ideal media for obtaining or purifying high amounts of virus in preparative applications such as viral vaccine production or virus purification, where they are already being satisfactorily applied [16–19]. The monolithic structure coupled with the QA chemistry, allowed us to obtain rotavirus recoveries of close to 100% with just a simple one-step elution at neutral pH using 1 M NaCl, which did not inhibit RT-qPCR. The convective flow generated within the monolithic material, in contrast with diffusive flow-based classic chromatography favours the rapid access of the viral particles to most of the monolith reactive surface and allows for operating at high flow rates [15]. Consequently, we found that rotavirus recovery was unaffected by increasing the flow rate from 2 to 6 ml/min (Tables 1 and 2). Furthermore, the scale up from CIM QA disk format to CIM QA 8 ml tube format, and the increase of the flow rate to 100 ml/min resulted in viral concentration rates of 56–66× from tap water samples (Table 3). This is close to the theoretical value of 65× expected when concentrating 1 l of water to 15 ml under 100% viral recovery conditions. The use of a 0.45 m pre-filter in the case of the river water samples resulted in approximately 2× decrease in the concentration rates but still allowed us to detect rotavirus concentrations as low as 97 particles/ml. At the flow rate used in the concentration experiments (100 ml/min) only 10 min were needed to concentrate 1 l of water. Taking into account that CIM QA 8 ml tubes can operate at flow rates of 400 ml/min and that increases in flow rate seem not to compromise viral recovery, using an appropriate pump 10 l of water could be processed in 25 min. Even when working with highly concentrated rotavirus loads, the flow-through fractions obtained when using QA monoliths were always rotavirus negative by RT-qPCR (Table 2). Such high viral binding efficiency together with the ability to work with high flow
rates may be exploited for other applications rather than merely concentration and detection such as, the removal of rotaviruses from potentially contaminated waters. The flexibility in terms of chemistry that CIM monoliths offer could be exploited by combining in tandem disposition CIM QA and CIM SO3 8 ml tubes in order to remove both negatively and positively charged viruses from water samples at environmental pH. 5. Conclusions CIM QA monolithic supports can efficiently bind rotaviruses present in stool samples as well as in tap and river water samples. Concentration of the rotaviruses is achieved by elution of bound viruses with 1 M NaCl. Eluted viruses preserve their integrity as verified under EM. The use of CIM QA 8 ml tubes allows for the processing of high water volumes in a reasonably short time depending on the flow rate capacity of the pump. The combination of concentration using CIM QA with detection using RT-qPCR resulted in the detection of rotavirus concentrations of as low as 97 particles/ml without the need of a secondary concentration step. The ability of CIM monoliths to bind viruses from large water volumes operating at high flow rates may be exploited for the removal of viruses from contaminated waters. Acknowledgement This work was supported by the Slovenian Research Agency and the Slovenian Ministry of Defense through the grant M1-0145 within the call for targeted research projects “Research for Security and Peace, 2006–2009, CRP-MIR”. References [1] M.K. Estes, in: D.M. Knipe, P.M. Howley, D.E. Griffin, R.A. Lamb, M.A. Martin, B. Roizman, S.E. Strais (Eds.), Fields Virology, 4th ed., Lippincot William and Wilkins, Philadelphia, PA, 2001, p. 1747. [2] M. Soriano-Gabarró, J. Mrukowicz, T. Vesikari, T. Verstraeten, Pediatr. Infect. Dis. J. 25 (2006) 7. [3] G.M. Schiff, G.M. Stefanovic, B. Yg, J.K. Pennekamp, in: J.L. Melnick (Ed.), Enteric Viruses in Water, vol. 5, Karger, Basel, 1984, p. 222. [4] C.N. Haas, J.B. Rose, C. Gerba, S. Regli, Risk Anal. 13 (1993) 545. [5] T.T. Fong, E.K. Lipp, Microbiol. Mol. Biol. Rev. 69 (2005) 357. [6] X.L. Pang, B. Lee, N. Boroumand, B. Leblanc, J.K. Preiksaitis, C.C. Yu Ip, J. Med. Virol. 72 (2004) 496. [7] P.H. Dennehy, D.R. Gauntlett, S.E. Spangenberger, J. Clin. Microbiol. 28 (1990) 1280. [8] I. Gutiérrez-Aguirre, A. Steyer, J. Boben, K. Gruden, M. Poljˇsak-Prijatelj, M. Ravnikar, J. Clin. Microbiol 46 (2008) 2547. [9] C. Gantzer, S. Senouci, A. Maul, Y. Levi, L. Schwartzbrod, Water Sci. Technol. 40 (1999) 105. [10] D.W. Griffin, K.A. Donaldson, J.H. Paul, J.B. Rose, Clin. Microbiol. Rev. 16 (2003) 129. [11] S. Jiang, R. Noble, W.P. Chui, Appl. Environ. Microbiol. 69 (2003) 179. [12] E.K. Lipp, J. Lukasik, J.B. Rose, Methods Microbiol. 30 (2001) 559. [13] R. Pallin, A.P. Wyn-Jones, B.M. Place, N.F. Lightfoot, J. Virol. Methods 67 (1997) 57. [14] J. Lukasik, T.M. Scott, D. Andryshak, S.R. Farrah, Appl. Environ. Microbiol. 66 (2000) 2914. ˇ [15] M. Barut, A. Podgornik, P. Brne, A. Strancar, J. Sep. Sci. 28 (2005) 1876. ˇ [16] F. Smrekar, M. Ciringer, M. Peterka, A. Podgornik, A. Strancar, J. Chromatogr. B 861 (2008) 177. [17] I. Kalashnikova, N. Ivanova, T. Tennikova, Anal. Chem. 80 (2008) 2188. ˇ [18] K. Branovic, D. Forcic, J. Ivancic, A. Strancar, M. Barut, T. Kosutic-Gulija, R. Zgorelec, R. Mazuran, J. Virol. Methods 110 (2003) 163. ˇ [19] P. Kramberger, N. Petroviˇc, A. Strancar, M. Ravnikar, J. Virol. Methods 120 (2004) 51. ˇ [20] J. Boben, P. Kramberger, N. Petroviˇc, K. Cankar, M. Peterka, A. Strancar, M. Ravnikar, Eur. J. Plant. Pathol. 118 (2007) 59. [21] Y.Z. Zheng, R. Webb, P.F. Greenfield, S. Reid, J. Virol. Methods 62 (1996) 153.
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Concentrating rotaviruses from water samples using monolithic chromatographic supports Ion Gutiérrez-Aguirre a,∗ , Marko Banjac b , Andrej Steyer c , Mateja Poljˇsak-Prijatelj c , b ˇ Matjaˇz Peterka b , Aleˇs Strancar , Maja Ravnikar a a b c
Department of Biotechnology and Systems Biology, National Institute of Biology, Veˇcna pot 111, SI-1000 Ljubljana, Slovenia BIA Separations, d.o.o., Teslova 30, SI-1000 Ljubljana, Slovenia Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Zaloˇska 4, SI-1000 Ljubljana, Slovenia
a r t i c l e
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Article history: Available online 6 November 2008 Keywords: Rotavirus Methacrylate monoliths Concentrating viruses Ion-exchange chromatography Water contamination RT q-PCR
a b s t r a c t Rotaviruses are the leading cause of diarrhoea in infants around the globe and, under certain conditions they can be present in drinking water sources and systems. Ingestion of 10–100 viral particles is enough to cause disease, emphasizing the need for sensitive diagnostic methods. In this study we have optimized the concentration of rotavirus particles using methacrylate monolithic chromatographic supports. Different surface chemistries and mobile phases were tested. A strong anion exchanger and phosphate buffer (pH 7) resulted in the highest recoveries after elution of the bound virus with 1 M NaCl. Using this approach, rotavirus particles spiked in 1 l volumes of tap or river water were efficiently concentrated. The developed concentration method in combination with a real time quantitative polymerase chain reaction assay detected rotavirus concentrations as low as 100 rotavirus particles/ml. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Rotaviruses are the main aetiological agent of acute viral gastroenteritis in young children around the globe [1]. They are responsible for 500 000–600 000 deaths each year, which primarily affect developing countries [2]. The infectious dose for rotaviruses, as for other enteric viruses, is extremely low, and as little as 10 viral particles can originate a gastroenteritis episode [3,4]. Rotaviruses are introduced in aquatic environments (river, lake, sea. . .), most commonly through leaking sewer systems, leaking septic systems, urban runoff and agricultural runoff [5]. Moreover rotavirus can contaminate drinking water supplies under defined conditions, such as, earthquakes and floods or intentional release. In such cases, undetectable but still infectious circulating concentrations of rotavirus constitute a risk to human health. Classic rotavirus detection methods consist of enzyme linked immunosorbent assays (ELISA), electron microscopy (EM) or polymerase chain reaction (PCR) [6,7]. Recently, a reverse transcription quantitative PCR (RT-qPCR) assay was developed, which offered improved sensitivity over other methods, detecting as low as 1 × 104 rotavirus particles/ml in river and tap water samples [8]. Still, for the detection of the low amounts of viruses expected
∗ Corresponding author. Tel.: +386 1 4233388; fax: +386 1 2573847. E-mail address: [email protected] (I. Gutiérrez-Aguirre). 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.10.106
in water samples, an initial concentration step has to be utilized. Methods used for concentrating viruses from water samples include: different filtration procedures based on the size of the virus (tangential flow filtration, vortex flow filtration) and/or adsorption–elution methods based on the surface charge of the virus (glass wool, positively or negatively charged filters) [9–13]. The concentrated fraction obtained with such methods has to be applied to a secondary concentration step which typically involves ultracentrifugation, further ultrafiltration or flocculation of the viruses. In this way 1–1000 l samples can be concentrated to 1–10 ml volumes and utilized for molecular or alternative detection techniques. Different concentration methods with their advantages and disadvantages are reviewed by Fong and Lipp [5]. The problems that arise when concentrating viruses from water samples usually depend on the method and include, high cost, unsuitability for large volumes, need for a secondary concentration step, clogging (filters), low recoveries, inhibitors transmitted to posterior molecular detection steps, etc. [5]. Because most enteric viruses are negatively charged at ambient pH, adsorption–elution with an electropositive filter is one of the most commonly used techniques [12], however electropositive filters are easily clogged and under defined conditions, such as marine water samples, they result on low recoveries [14]. In this work we used methacrylate monoliths to bind and concentrate rotaviruses from water samples. Methacrylate mono-
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liths, commercialized by BIA Separations under the name CIM (convective interaction media), are made of a single block of highly porous polymer, where the mass transfer, unlike traditional beadbased chromatography, is based on convective flow, allowing a more rapid and efficient exchange between mobile and stationary phases [15]. Furthermore, the large diameter (up to 1500 nm) of the highly interconnected channels integrating the monoliths favours the circulation and interaction with large biomolecules such as viruses [15]. The methacrylate monoliths can bear different ligands on the surface allowing their use for anionic/cationic exchange, affinity, and hydrophobic interaction chromatography [15]. CIM monoliths have already been successfully applied for the binding, separation and/or concentration of different viruses, such as bacteriophage T4 [16], influenza [17], measles and mumps [18] and tomato mosaic virus (ToMV) [19,20]. In order to optimize the binding of rotaviruses to CIM, different anion-exchange (QA) and cation-exchange (SO3 ) chemistries, as well as different pH and elution conditions were tested using disk format (bed volume 0.34 ml). The chromatographic fractions were characterized by EM and ELISA, and the viral concentration in each fraction was quantified by RT-qPCR. The optimized conditions were used in concentration experiments using artificially inoculated 1 l tap or river water samples applied to CIM tube format (bed volume 8 ml).
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hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffer pH 7, 50 mM phosphate buffer pH 7, 50 mM acetate buffer pH 4 and 50 mM citrate buffer pH 9. Different NaCl concentrations used in linear gradient as well as stepwise elutions are indicated in the figures and tables. The loaded samples were always prepared from rotavirus positive clarified 10% stool suspensions of known concentration estimated by counting under EM [21]. In order to dilute the traces of NaCl inherent to the clarified stool, which could interfere with the virus binding to the ion-exchange support, the initial sample was always diluted at least 10-fold in the mobile phase (buffer A) before loading to the CIM disk. All load, flow through and elution fractions were characterized with EM and ELISA, and rotavirus amount was quantified with RT-qPCR as described below. To calculate the capacity of the QA monolith to bind rotavirus, a stool suspension of known rotavirus concentration was continuously loaded into a CIM QA disk at 6.2 ml/min. Flow through was collected in 10 ml fractions, and fractions were applied to rotavirus specific ELISA detection. The first ELISA positive fraction was considered as 0% breakthrough, and the capacity of rotavirus particles/ml of monolithic support was calculated accordingly. CIM monolithic columns were regenerated and stored as recommended by the manufacturer. 2.3. Quantification of rotavirus in chromatographic fractions by RT-qPCR
2. Experimental 2.1. Characterization of rotavirus positive stool samples and fractions: EM and ELISA Rotavirus positive stool samples were obtained from children hospitalized with acute gastroenteritis. Samples were characterized, clarified and 10% stool suspensions prepared as described by Gutiérrez-Aguirre et al. [8]. The concentration of rotavirus in the initial 10% stool suspension was estimated by virus counting under an electron microscope JEM 1200 EXII (Jeol, Tokyo, Japan) using the latex-negative staining technique [21]. Such samples were used for preparing the loads used in the optimizations of the rotavirus binding to CIM, and for inoculating the tap and river water samples used in concentration experiments. EM was also used to evaluate the integrity of the viruses eluted from the CIM monolithic columns. A rotavirus specific ELISA assay (Premier Rotaclone, Meridian Bioscience, Cincinnati, OH, USA) was used for fast monitoring of chromatographic fractions for rotavirus presence. 2.2. Optimization of the rotavirus binding to CIM Strong anion- and cation-exchange CIM disk monolithic columns (BIA Separations, Ljubljana, Slovenia) with a bed volume of 0.34 ml, a diameter of 12 mm and a length of 3 mm, were used to optimize the virus binding to the support as well as the posterior elution. As anion exchanger the positively charged quaternary amine (QA) surface chemistry was chosen, while as cation exchanger negatively charged sulfuric anhydride (SO3 ) was utilized. The disks were placed in a specially designed housing (BIA Separations, Ljubljana, Slovenia) and connected to a gradient HPLC system (Knauer, Berlin, Germany). The system was composed of two K-500 pumps, a UV–vis detector K-2500 operating at 280 nm with a 10 mm optical path and 10 l volume flow cell connected by 0.25 mm i.d. polyether ether ketone (PEEK) capillary tubes. An in-line conductivity monitor (Amersham Biosciences, Uppsala, Sweden) was used to monitor the ionic strength of the mobile phase. HPLC software from Knauer was used in all experiments. Unless otherwise indicated, flow rate was set to 2 ml/min. Different mobile phases were tested: 50 mM (4-(2-
For the quantification of the rotavirus amount present in each chromatographic fraction a recently developed RT-qPCR was used [8]. In all experiments RNA from the known-concentration stool suspension (used for load preparation), was isolated and 10-fold serially diluted from neat to 1 × 10−6 . Each dilution was then applied to rotavirus specific RT-qPCR in triplicate, and threshold cycles (PCR cycle in which the real time amplification curve becomes exponential = Ct) were calculated, in order to build a standard curve of log(concentration) vs Ct [8]. In addition, the RNA from the fractions generated after each chromatographic run (load—L, flow through—FT, elution 1—E1, elution 2—E2) was also isolated and applied in triplicate to rotavirus specific RT-qPCR. The Ct obtained for each fraction was used to estimate the rotavirus concentration via the previously generated standard curve. For the concentration experiments in tap and river water samples, known concentration (2 ng) of luciferase RNA was added to the samples as an external control immediately prior to the RNA isolation. Each RNA dilution was then applied not only to rotavirus specific RT-qPCR but also to luciferase specific RT-qPCR [8]. The Ct obtained for luciferase were used to normalize the rotavirus specific Ct, to account for variations due to other factors (rather than solely the virus concentration), such as, differences in RNA extraction yield or presence of PCR inhibitors. In all experiments RNA was isolated from 250 l of sample using TRIzol reagent, according to manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA) 2.4. Concentration experiments A rotavirus positive stool suspension of known concentration was used as seed to inoculate samples consisting of 1 l of tap or river water to different final concentrations. These samples were pumped through a CIM QA 8 ml tube column using a Milton Roy LMI B71 dosing pump (Milton Roy Europe, Point Saint Pierre, France) operating at 100 ml/min flow rate. At least 10 column volumes (CVs) of 50 mM phosphate pH 7 were used to wash any unbound compound from the CIM QA tube after the loading, and then the bound viruses were eluted by using 1 M NaCl in the same buffer. Elution fraction volume was always between 10 and 15 ml. The elution was monitored spectrophotometrically at 254 nm with a Smartline
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preparative UV Detector 200 (Knauer) equipped with a preparative flow cell. All connections were made using HPLC grade PEEK tubing of appropriate i.d. for preparative applications and corresponding fittings. In the case of river water samples a 0.45 m cut off–142 mm i.d. filter (Sartorius, Goettingen, Germany) in a specially designed metallic housing, was placed between the pump and the CIM QA tube. Samples before and after concentration were applied to RT-qPCR detection and quantification as described above, and concentration factors were calculated. For detection purposes, samples giving a positive Ct value in the rotavirus specific RT-qPCR in at least two of the three replicates were considered as rotavirus positive. Eight ml CIM tube monolithic columns were regenerated and stored as recommended by the manufacturer. 3. Results 3.1. Binding of rotavirus to CIM monolithic columns First, we tested the binding of the virus at neutral pH (pH 7) using a strong anion exchanger (QA) and a strong cation exchanger (SO3 ). Four ml of a stool sample (1.64 × 1010 particles/ml of G2P [4] rotavirus) 10-fold diluted in buffer A (HEPES 50 mM pH 7) were loaded on both disk QA and SO3 monolithic columns, and eluted with a gradient of 0–2 M NaCl in buffer A (Fig. 1). In the case of the QA disk, rotaviruses were detected by ELISA only in the elution peak (E1), and not in the flow through (FT), while the opposite occurred when using SO3 chemistry (Fig. 1). Therefore one can conclude that rotaviruses bind efficiently to CIM QA monoliths, as it was expected taking into account that most enteric viruses are negatively charged at ambient pH [12]. From the gradient shown in the upper panel of Fig. 1, it seems that most of the bound material is eluted at NaCl concentrations between 0.1 and 0.3 M NaCl. In order to obtain a direct proof of the integrity of the eluted virus, FT and E1 fractions were characterized by EM, and the intact rotavirus particles were observed indicating no change in their shape and structure as they pass through the monolithic support (Fig. 1, lower panel). Next, we decided to determine the capacity of the QA monolithic support to bind rotaviruses present in a real stool sample. A rotavirus stool of 2.66 × 1010 particles/ml was continuously loaded into a CIM QA disk and the flow through was collected in 10 ml fractions, which were applied to rotavirus detection by ELISA. The first rotavirus positive fraction was considered as the 0% breakthrough of the capacity, and this occurred in the sixth fraction indicating that we were able to load 50 ml of the stool without going beyond capacity. This resulted in a capacity estimation of 4 × 1012 rotavirus particles/ml of CIM QA monolith for a real stool suspension at 0% breakthrough. 3.2. Optimization of rotavirus recovery from QA monoliths Prior to concentration experiments using higher water volumes, conditions, which enable the highest viral recoveries needed to be determined, i.e., mobile phase buffer composition, pH and NaCl concentration in elution buffer. Different runs with different conditions were performed (Table 1). In all cases the runs consisted of a FT followed by two step gradient elutions (Fig. 2). The first elution peak (E1) was obtained by eluting with 0.6, 0.8 or 1 M NaCl concentration in buffer A, while the second elution peak (E2) was always obtained by eluting with 2 M NaCl (in order to release the material that remained attached to CIM after first elution, see Table 1). The amount of rotavirus present in the loaded sample as well as in FT, E1 and E2 fractions was quantified using RT-qPCR, and percentages of viral recovery were calculated (Table 2). In all the tested runs the amount of rotavirus in the FT fractions was 0% (Table 2) indicating that under all conditions CIM QA is able to efficiently
Fig. 1. Chromatographic runs on disk CIM QA and CIM SO3 monolithic columns. (Upper panel) A stool containing rotavirus G2P [4], as estimated by EM counting, at 1.64 × 1010 particles/ml was diluted 10-fold in buffer A (HEPES 50 mM, pH 7) and 4 ml loaded into a disk CIM QA monolithic column. Flow rate was kept at 2 ml/min. After a wash step using buffer A, a linear gradient 0–100% of buffer B (HEPES 50 mM, NaCl 2 M, pH 7) was applied. Flow through (FT) and elution peaks (E1) are indicated in the panel. Both FT and E1 were applied to rotavirus specific ELISA detection and results (+ or −) are indicated in the panel. The thick line represents A280 , while the thin line represents the conductivity. (Middle panel) Same as in upper panel except the disk monolithic column used, which in this case was SO3 . (Lower panel) Electron microscope micrograph of one of the fractions corresponding to the E1 peak in the upper panel.
bind the viruses. The viral recoveries obtained in the first elution peak varied depending on the NaCl concentration used, namely 40%, 86%, and 91% of the loaded virus was recovered when eluting with 0.6, 0.8 and 1 M NaCl, respectively (Tables 1 and 2, runs 1–3). In runs 4 and 5, we evaluated the effect on the recovery of using basic and acid pH in the mobile phase. The use of sodium acetate buffer pH 4 and sodium citrate buffer pH 9 resulted in a 30% and 15% decrease, respectively, of the recovery in comparison to HEPES buffer pH 7 (Tables 1 and 2, runs 3–5). In the last run
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Table 1 Run conditions tested in disk CIM QA columns. Conditions Run
Loada
Flow rate
Buffer A
pH
[NaCl] Bb
[NaCl] C2
1 2 3 4 5 6
4 ml 4 ml 4 ml 4 ml 4 ml 10 ml
2 ml/min 2 ml/min 2 ml/min 2 ml/min 2 ml/min 6 ml/min
HEPES 50 mM HEPES 50 mM HEPES 50 mM Acetate 50 mM Citrate 50 mM Phosphate 50 mM
7 7 7 4 9 7
0.6 M 0.8 M 1M 1M 1M 1M
2M 2M 2M 2M 2M 2M
a Load was in all cases prepared by 10× diluting in the corresponding buffer A, a stool suspension containing rotavirus G2P [4] at 1.64 × 1010 particles/ml. b Concentration of NaCl included in buffer A in order to get buffer B (first elution step) and buffer C (second elution step).
Table 2 Rotavirus recoveries from the chromatographic runs listed in Table 1. Run
1 2 3 4 5 6
Fig. 2. Typical rotavirus binding and step gradient elution using disk CIM QA monolithic column. Run conditions and virus recoveries are those shown for run number 6 in Tables 1 and 2.
Virus recovery (%) FT
E1
E2
Total
0 0 0 0 0 0
40.7 86.8 91.1 58 76 99.8
13.3 1.48 0.8 3.8 0.7 2.2
54 88.3 92 61.8 76.7 102
(run 6) the buffer was changed to phosphate pH 7, and the load and flow rate were increased to 10 ml and 6 ml/min, respectively. These changes improved recovery of the virus at values close to 100% in the first elution peak (Tables 1 and 2, run 6) (Fig. 2). Therefore, in following experiments with higher volume samples we used run 6 conditions: phosphate buffer 50 mM, pH 7 and an elution step using 1 M NaCl. 3.3. Concentration of rotavirus from spiked tap and river water samples To determine the ability of CIM QA monoliths to concentrate low numbers of rotaviruses present in tap or river waters, we inoculated 1 l samples of the mentioned waters to different rotavirus concentrations. The HPLC system and the CIM QA disk monolithic columns were substituted by a dosage membrane pump operating at 100 ml/min, a modular preparative UV detector and CIM QA 8 ml tube monolithic columns. A 0.45 m pore size filter was placed between the pump and the column in the cases when river water was tested to prevent larger particles from getting into the col-
umn. The pH of the water samples was adjusted by adding 500 mM phosphate buffer, pH 7, in a 1:10 (v/v) ratio. All the load and elution fractions were applied to RT-qPCR detection and quantification and where applicable, concentration factors were estimated (Table 3). The volume of the elution peak was in all the cases close to 15 ml meaning that if starting from a 1000 ml sample, with a 100% effective concentration, a concentration factor of 65 times would be expected. Out of the three tap water samples tested before the concentration step, rotaviruses were detected in two (9.7 × 106 and 9.7 × 103 particles/ml). RT-qPCR was unable to detect viruses in the tap water of the third sample seeded to a final concentration of 97 particles/ml; this was only possible after the concentration using CIM tube. For the first two samples we were able to calculate concentration factors, which ranged from 56 to 66× (Table 3), indicating a very efficient viral recovery, as it was already observed when working with CIM QA disk format. Concerning river water samples, RT-qPCR could detect rotavirus down to 9.7 × 102 particles/ml, coinciding with the detection limit of the method, Ct ≈ 37 [8] (Table 3). After concentration using CIM QA tube we were able to confidently detect both 9.7 × 102 and 97 particles/ml. The calculated concentration factors ranged from 20 to 40× (Table 3). The decrease in the concentration yield in comparison with tap water is most probably due to the presence of the in-line filter which in contrast to tap water experiments generates a surface where a portion of the viruses can unspecifically adsorb. Still, the procedure was enough efficient to enable the detection of samples, which were otherwise beyond the limit of detection of RT-qPCR.
Table 3 Concentration of rotaviruses spiked in 1-l samples of tap and river water. Spike [RoV] (particles/ml)
Ct load
Ct concentrate
Concentration factora
Tap water
9.7 × 10 9.7 × 103 9.7 × 101 Non-spiked
23.9 (+) 34.1 (+) Non-detected Non-detected
19.1 (+) 28.4 (+) 33.6 (+) Non-detected
66× 56× nmb nm
River water
9.7 × 106 9.7 × 105 9.7 × 104 9.7 × 103 9.7 × 102 9.7 × 101 Non-spiked
24.5 (+) 27.8 (+) 32 (+) 34.6 (+) 37.7 (+) Non-detected Non-detected
19.2 (+) 19.6 (+) 23.5 (+) 26.7 (+) 35.8 (+) 36.6 (+) Non-detected
22× 40× 40× 20× nm nm nm
6
a b
Concentration factor was calculated using RT-qPCR for quantifying the virus concentration both the load and the concentrate as indicated in Section 2. Non-measurable. Concentration factor for samples giving Ct values higher than 35 (beyond limit of quantification) was not measured.
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4. Discussion The objective of our study was to use CIM chromatographic supports for fast and efficient concentration of rotaviruses that, under specific circumstances, are present in tap and environmental water samples. Classic concentration methods consist mainly of the use of ultrafiltration units, glass wool, polyethyleneglycol (PEG) precipitation, ultracentrifugation, flocculation and binding to charged membranes [9–13]. Lately, the use of charged membranes is becoming one of the most utilized methods for concentration of viruses in water samples [5]. Adsorption–elution to electropositive membranes is, for example, the designated method for concentration of enteric viruses from drinking waters by the Information Collection Rule of the US Environmental Protection Agency (http://www.epa.gov/nerlcwww/icrmicro.pdf). The relative simplicity of their use and the ability to process large water volumes are among the advantages of such membranes [5]. Low recoveries in some cases, use of elution buffers with components that can inhibit posterior PCR detection (high pH + beef extract) and need of an additional secondary concentration are among the disadvantages [5]. In this work we took advantage of the properties of the CIM monolithic supports and apply them to the concentration of rotaviruses from different water samples. The highly porous (macropores and mesopores) structure of the CIM monolithic support provides a high surface area accessible to the viral particles resulting in very high binding capacities and recoveries [15]. We obtained a very high binding capacity of 4 × 1012 rotavirus particles/ml of CIM QA monolith for a clarified stool suspension at 0% breakthrough, therefore, as much as 3.2 × 1013 particles of rotaviruses could be accommodated into a CIM 8 ml QA tube. Such high viral binding capacities make CIM monoliths ideal media for obtaining or purifying high amounts of virus in preparative applications such as viral vaccine production or virus purification, where they are already being satisfactorily applied [16–19]. The monolithic structure coupled with the QA chemistry, allowed us to obtain rotavirus recoveries of close to 100% with just a simple one-step elution at neutral pH using 1 M NaCl, which did not inhibit RT-qPCR. The convective flow generated within the monolithic material, in contrast with diffusive flow-based classic chromatography favours the rapid access of the viral particles to most of the monolith reactive surface and allows for operating at high flow rates [15]. Consequently, we found that rotavirus recovery was unaffected by increasing the flow rate from 2 to 6 ml/min (Tables 1 and 2). Furthermore, the scale up from CIM QA disk format to CIM QA 8 ml tube format, and the increase of the flow rate to 100 ml/min resulted in viral concentration rates of 56–66× from tap water samples (Table 3). This is close to the theoretical value of 65× expected when concentrating 1 l of water to 15 ml under 100% viral recovery conditions. The use of a 0.45 m pre-filter in the case of the river water samples resulted in approximately 2× decrease in the concentration rates but still allowed us to detect rotavirus concentrations as low as 97 particles/ml. At the flow rate used in the concentration experiments (100 ml/min) only 10 min were needed to concentrate 1 l of water. Taking into account that CIM QA 8 ml tubes can operate at flow rates of 400 ml/min and that increases in flow rate seem not to compromise viral recovery, using an appropriate pump 10 l of water could be processed in 25 min. Even when working with highly concentrated rotavirus loads, the flow-through fractions obtained when using QA monoliths were always rotavirus negative by RT-qPCR (Table 2). Such high viral binding efficiency together with the ability to work with high flow
rates may be exploited for other applications rather than merely concentration and detection such as, the removal of rotaviruses from potentially contaminated waters. The flexibility in terms of chemistry that CIM monoliths offer could be exploited by combining in tandem disposition CIM QA and CIM SO3 8 ml tubes in order to remove both negatively and positively charged viruses from water samples at environmental pH. 5. Conclusions CIM QA monolithic supports can efficiently bind rotaviruses present in stool samples as well as in tap and river water samples. Concentration of the rotaviruses is achieved by elution of bound viruses with 1 M NaCl. Eluted viruses preserve their integrity as verified under EM. The use of CIM QA 8 ml tubes allows for the processing of high water volumes in a reasonably short time depending on the flow rate capacity of the pump. The combination of concentration using CIM QA with detection using RT-qPCR resulted in the detection of rotavirus concentrations of as low as 97 particles/ml without the need of a secondary concentration step. The ability of CIM monoliths to bind viruses from large water volumes operating at high flow rates may be exploited for the removal of viruses from contaminated waters. Acknowledgement This work was supported by the Slovenian Research Agency and the Slovenian Ministry of Defense through the grant M1-0145 within the call for targeted research projects “Research for Security and Peace, 2006–2009, CRP-MIR”. References [1] M.K. Estes, in: D.M. Knipe, P.M. Howley, D.E. Griffin, R.A. Lamb, M.A. Martin, B. Roizman, S.E. Strais (Eds.), Fields Virology, 4th ed., Lippincot William and Wilkins, Philadelphia, PA, 2001, p. 1747. [2] M. Soriano-Gabarró, J. Mrukowicz, T. Vesikari, T. Verstraeten, Pediatr. Infect. Dis. J. 25 (2006) 7. [3] G.M. Schiff, G.M. Stefanovic, B. Yg, J.K. Pennekamp, in: J.L. Melnick (Ed.), Enteric Viruses in Water, vol. 5, Karger, Basel, 1984, p. 222. [4] C.N. Haas, J.B. Rose, C. Gerba, S. Regli, Risk Anal. 13 (1993) 545. [5] T.T. Fong, E.K. Lipp, Microbiol. Mol. Biol. Rev. 69 (2005) 357. [6] X.L. Pang, B. Lee, N. Boroumand, B. Leblanc, J.K. Preiksaitis, C.C. Yu Ip, J. Med. Virol. 72 (2004) 496. [7] P.H. Dennehy, D.R. Gauntlett, S.E. Spangenberger, J. Clin. Microbiol. 28 (1990) 1280. [8] I. Gutiérrez-Aguirre, A. Steyer, J. Boben, K. Gruden, M. Poljˇsak-Prijatelj, M. Ravnikar, J. Clin. Microbiol 46 (2008) 2547. [9] C. Gantzer, S. Senouci, A. Maul, Y. Levi, L. Schwartzbrod, Water Sci. Technol. 40 (1999) 105. [10] D.W. Griffin, K.A. Donaldson, J.H. Paul, J.B. Rose, Clin. Microbiol. Rev. 16 (2003) 129. [11] S. Jiang, R. Noble, W.P. Chui, Appl. Environ. Microbiol. 69 (2003) 179. [12] E.K. Lipp, J. Lukasik, J.B. Rose, Methods Microbiol. 30 (2001) 559. [13] R. Pallin, A.P. Wyn-Jones, B.M. Place, N.F. Lightfoot, J. Virol. Methods 67 (1997) 57. [14] J. Lukasik, T.M. Scott, D. Andryshak, S.R. Farrah, Appl. Environ. Microbiol. 66 (2000) 2914. ˇ [15] M. Barut, A. Podgornik, P. Brne, A. Strancar, J. Sep. Sci. 28 (2005) 1876. ˇ [16] F. Smrekar, M. Ciringer, M. Peterka, A. Podgornik, A. Strancar, J. Chromatogr. B 861 (2008) 177. [17] I. Kalashnikova, N. Ivanova, T. Tennikova, Anal. Chem. 80 (2008) 2188. ˇ [18] K. Branovic, D. Forcic, J. Ivancic, A. Strancar, M. Barut, T. Kosutic-Gulija, R. Zgorelec, R. Mazuran, J. Virol. Methods 110 (2003) 163. ˇ [19] P. Kramberger, N. Petroviˇc, A. Strancar, M. Ravnikar, J. Virol. Methods 120 (2004) 51. ˇ [20] J. Boben, P. Kramberger, N. Petroviˇc, K. Cankar, M. Peterka, A. Strancar, M. Ravnikar, Eur. J. Plant. Pathol. 118 (2007) 59. [21] Y.Z. Zheng, R. Webb, P.F. Greenfield, S. Reid, J. Virol. Methods 62 (1996) 153.