Development of a Novel Filter Cartridge System with Electropositive

May 27, 2014 - To evaluate the system's efficiency in viral recovery, poliovirus (PV-1), a surrogate for enteric viruses, was used to artificially con...
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Development of a Novel Filter Cartridge System with Electropositive Granule Media to Concentrate Viruses from Large Volumes of Natural Surface Water Min Jin,† Xuan Guo,† Xin-Wei Wang, Dong Yang, Zhi-Qiang Shen, Zhi-Gang Qiu, Zhao-Li Chen, and Jun-Wen Li* Department of Environment and Health, Institute of Health and Environmental Medicine, Key Laboratory of Risk Assessment and Control for Environment & Food Safety, Tianjin 300050, China S Supporting Information *

ABSTRACT: Exposure to various infectious viruses in environmental drinking water can constitute a public health risk. However, it is difficult to detect viruses in water due to their low concentration. In this study, we have developed a novel filter cartridge system containing electropositive granule media (EGM). Viruses present in large volumes of environmental samples were adsorbed onto the EGM, and then recovered by elution and poly(ethylene glycol) (PEG) concentration. To evaluate the system’s efficiency in viral recovery, poliovirus (PV-1), a surrogate for enteric viruses, was used to artificially contaminate river water samples which were then assayed by quantitative real-time PCR. To optimize the concentration procedure, the eluent type, water flow rate and properties (e.g., pH, bacterial, and viral loads), were evaluated. The highest virus recovery was obtained by pumping river water at a flow rate of 300 mL/min and then pushing 3 L of an eluent containing 3× broth [1.5% (w/v) NaCl, 3% (w/v) tryptone, 1.5% (w/v) beef powder] with 0.05 mol/L glycine through the filter. Using this procedure, the recovery efficiencies of PV-1 from 10 to 100 L of spiked river water were up to 99%. In addition, this method is virus load and pH dependent. Virus recovery was maximal at a load of between 103.5 and 105.5 TCID50 and a pH ranging from 5 to 7. The bacterial load in the water has no effect on virus recovery. Different types of viruses and surface water were tested to validate the system’s applicability. Results revealed that the EGM filter cartridge was able to concentrate PV-1, human adenoviruses (HAdVs) and noroviruses (HuNoVs) with high efficiency from river, lake, and reservoir water. Furthermore, it showed more efficient recovery than glass wool and 1MDS filters. These data suggest that this system provides rapid and efficient virus recovery from a large volume of natural surface water and, as such, could be a useful tool in revealing the presence of viruses in surface water. determine the epidemiology of circulating enteric viruses,11 and to better control waterborne outbreaks caused by viruses.12−17 However, due to the low amounts of viral particles present in natural surface water [ 0.05, Figure 6a). In addition, the EGM filter cartridge system had low bacterial recovery. Less than 20% of the input E. coli was recovered from both the first-stage elution solution and final buffered virus concentrate (SI Figure S10). Furthermore, virus recovery was investigated at different pH values of river water by adjusting the pH between 3.0 and 10.0 using 1 mol/L HCl or NaOH. Similar to the pH dependence of other virus concentration techniques relying on electronegative and electropositive media,34,37 the recovery efficiency of the cartridge system with EGM was significantly affected by the pH of the water. Our results show that virus recovery was maximal at a pH range of between 5 to 7 (p > 0.05, Figure 6b). Virus recovery declined substantially with increasing or decreasing pH, reaching a value of less than 10% at pH 10 and pH 3. Validation of Virus Concentration using the Filter Cartridge with EGM. To further validate the applicability of the filter cartridge system with EGM, 20 L samples of surface water sourced from the Haihe river (n = 3), Jinhe river (n = 3), Tuanbo lake (n = 3) and Yunqiao reservoir (n = 3) were spiked with 1 × 106.5 TCID50 of PV-1 [(1.8 ± 0.37) × 108 GCs],

Figure 4. Recovery of spiked PV-1 from 20 L of river water using the filter cartridge under different water flow rates and assayed by RTqPCR (n = 3). No environmental/background HRV were found and the mean concentration of background PV-1 in the tested water was calculated as 80 GCs/L by RT-qPCR.

between the recovery yields of PV-1 under different water flow rates (p < 0.05). An average of 71% PV-1 recovery was obtained at a water flow rate of 300 mL/min. Once the water flow rate was over 500 mL/min, less than 50% of viruses were recovered. Virus Recovery from 10 L, 20 or 100 L River Water Samples Spiked with Different Virus Concentrations by the Filter Cartridge with EGM. Figure 5 shows the recovery yields of PV-1 assayed by RT-qPCR from 10 L, 20 L, and 100 L of river water spiked with approximately 103.5−109.5 TCID50 of PV-1 [(7.9 ± 2.47) × 105 GCs − (8.0 ± 1.63) × 1010 GCs] using the filter cartridge with EGM (flow rate of 300 mL/min). It shows that viral inputs clearly affect the recovery yields, but there were no significant differences between virus recoveries from different river volumes with the same virus input. When PV-1 inputs ranged from 103.5 to 105.5 TCID50 [(7.9 ± 2.47) × 6952

dx.doi.org/10.1021/es501415m | Environ. Sci. Technol. 2014, 48, 6947−6956

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Figure 6. Virus Recovery from 20 L of river water using the filter cartridge under different E. coli concentrations (a) and pH (b) conditions and assayed by RT-qPCR (n = 3). Approximately 106.5 TCID50 of PV-1[(1.9 ± 0.95) × 108 GCs)] was spiked into the river water samples. No environmental/background HRV were found and the mean concentration of background PV-1 in the tested water was calculated as 3.9 × 103 GCs/L by RT-qPCR. The mean values for total bacteria (TB) and total coliform (TC) in the river water (background) were 2300 and 300 CFU/mL, respectively.

HAdV 41 [(3.7 ± 1.26) × 108 GCs] and of HuNoV GII [ (2.7 ± 0.21) × 106 GCs], respectively. The samples were then used to determine virus recovery from the filter cartridge system. Although viruses differ in their electronegativity and, therefore, in their binding efficiency to electropositive filters, the results demonstrated that the filter cartridge system with EGM had an excellent capacity to concentrate viruses from natural surface water. Each virus tested could be recovered from the four water types at a high efficiency. Average virus recovery was over 87% (SI Table S1). Comparative Study of the Virus Concentration Methods. PV-1 recoveries from the 20 L of spiked river water samples using the filter cartridge with EGM were compared with those using the glass wool filters and the 1MDS filters. The results demonstrated that the filter cartridge with EGM showed almost perfect adsorption of PV-1 and that its average recovery of PV-1 from river water reached 99% from an input of 103.8 TCID50-104.8 TCID50 [(7.8 ± 1.52) × 105 GCs − (6.3 ± 1.07) × 107 GCs] whereas the glass wool and the 1MDS filters showed less than 50% virus recovery, respectively. The mean recoveries of each concentration are presented in SI Table S2. Therefore, virus recovery using the filter cartridge with EGM was the highest, resulting in a statistically significant difference between the other two concentration methods (p < 0.05).

Viruses will be positively charged below their isoelectric point (IEPs), and negatively charged above the IEP. Generally, the IEPs of viruses are within the 1.9−8.4 pH range and so, the net electrostatic charge of most viruses such as PV-1 will be negative45 at the pH of natural surface water, which ranges from 6 to 9. The IEP of Al(OH)3 precipitate is between 8.5 and 9.3, and so the EGM will be positively charged at pH 6−9 facilitating the absorption and concentration of negatively charged viruses from water. For the same reasons, the EGM surface would become negatively charged on exposure to a solution at high pH, creating unfavorable conditions for viruses to absorb. However, in practice, this is not the case because there are still many other factors that limit virus recovery including the physicochemical properties of the surrounding aqueous environment (e.g., ionic strength), and the presence of organic compounds.25 In this study, several other important factors were found to contribute to the high virus recovery of the EGM filter. First, it is the fundamental to have a high virus binding capacity. The binding capacity of the filter depends on the negative or positive charge loads on the media surface, which is associated with the specific surface area and zeta potential of electropositive media under a given condition. In this study, the EGM has an extensive surface area (500 m2/g) compared with glass wool and Zeta Plus Virosorb 1MDS. It also possessed a strongly positive electrophoretic mobility at a pH ranging from 5 to 8 and showed strong virus binding characteristics in that pH range. Correspondingly, the high surface area coupled with the relatively high isoelectric point (IEP range = 8.5 to 9.3) confers a strong electropositivity and binding capacity to the filter surface, which is higher than that of glass wool and Zeta Plus Virosorb 1MDS. Second, the water flow rate, which determines the contact time between viruses and cations on the EGM, is also a key parameter determining virus recovery. Generally, with an increase in water flow rate, virus recovery decreases. We observed that the optimal flow rate of 300 mL/ min resulted in high recovery efficiencies using this method. 1MDS and NanoCeram filters use increased flow rates to prevent clogging (an average flow rate of 5.6 L/min), which can result in low virus recovery.36,46 Finally, the biological properties of water, particular viral loads, had a notable effect on virus recovery. For example, the filter cartridge in this study was filled with 600 g of EGM, and thus the positive charge load

4. DISCUSSION In this study, we have developed a novel and convenient filter cartridge system with EGM to concentrate viruses from large volumes of natural surface water even at high turbidities (10− 32 NTU). Although the values of viral recoveries vary for the different conditions, this study demonstrates that high recoveries of above 99% were achieved for 10 3.5−10 5.5 TCID50 or 7.9 × 105 − 4.9 × 107 GCs of virus load from 10 to 100 L of natural surface water and that this method was more effective than others published elsewhere15,26,33,36,44 (SI Table S3). Importantly, the filter cartridge with EGM requires no clarification of surface water, and so viruses in river water that adsorbed onto solid matrices by electrostatic interactions were also recovered by alkaline elution which may result in higher virus recovery than other reported methods. In addition, the adsorption of bacteria present in the water samples does not affect virus recovery. 6953

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Information. This material is available free of charge via the Internet at http://pubs.acs.org/.

on the media surface (i.e., the binding capacity for virus), was limited. Once the charged media was saturated with viruses or other negative particles, no more virus absorption occurred and virus recovery decreased accordingly. Our results suggest that high virus recovery of 99% was achieved with the filter cartridge from 10 up to 100 L of river samples spiked with below 105.5 TCID50 PV-1 (4.9 × 107 GCs). If the PV-1 load was too high (i.e., 108.5 TCID50) and beyond the binding capacity of the filter, virus recovery declined drastically to less than 10%. Fortunately, because the concentration of viruses in natural surface water are not generally above 104 pfu/L or 106 GCs/ L,18−23 the filter cartridge with EGM should be able to adequately concentrate viruses from large volumes of natural surface water (10−100 L). During this study, it was also found that samples processed from large volumes of water tended to cause inhibitory effects more frequently than those from smaller volumes. This indicates that some substances, for example, humic and fulvic acids from environmental samples, coconcentrated with the viruses caused detection inhibition.47,48 In addition, the beef extract used in one of the eluates contains high concentrations of poorly characterized components, which may interfere with qPCR and contribute to the inhibition of qPCR in detecting viruses.49 Three effective approaches were taken in this study to avoid these inhibitory elements. First, a two-step RT-qPCR format was used. It was observed that inhibition was a major problem for PV-1 detection in the river water, as described previously, but much less problematic for HAdV-41 detection.50,51 Therefore, we believe that the inhibition occurred during the RT phase (i.e., only used for PV-1). In addition, in the one-step RT-qPCR detection assay, it was found that inhibitors present in the extracted RNA significantly affected the fluorescent signal produced as the amplification curves were “jagged” even when the extracted RNA from the water samples was diluted 1:10. This phenomenon was not observed during the two-step RT-qPCR since the inhibitors had been diluted out during the qPCR step (SI Figure S11). Second, internal controls were incorporated in the assays to evaluate inhibition problems during qRT-PCR as previously done by other groups.3,50,52−54 By using internal controls, all nucleic acid extracts were amplified to verify the impact of inhibitors, both in undiluted and diluted form (up to 100-fold). Third, PEG precipitation following elution was used to remove partial inhibitors, as previously described.26,55 In conclusion, we have developed a new filter cartridge system with EGM for the recovery of dilute viruses from largevolumes of natural surface water samples. Not only have we shown high virus recovery from river, lake and reservoir water, but also describe a system without the need for pretreatments such as clarification. Due to the high efficiency of the virus concentration step, this new system should improve the detection rate of various viruses and has sufficient potential to be applied to the detection of viruses from natural surface water. It should also be effective for other types of freshwater, including tap, recreational and groundwater.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-22-84655345; fax: +86-22-23328809; e-mail: [email protected]. Author Contributions †

M.J. and X.G. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by grants from the National Natural Science Foundation of China (81372947, 30930078, 81100100), National High Technology Research and Development Program of China (2009AA06Z404).

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ABBREVIATIONS: T water temperature TC total coliforms TB total bacteria TTC thermotolerant coliforms NTU nephelometric turbidity unit

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ASSOCIATED CONTENT

S Supporting Information *

We present details for microorganisms and cell lines, quality of water samples, filter cartridge binding capacity, eluents constitute, concentration process of glass wool filter and a 1MDS filter, reaction condition of RT-qPCR assays and some results of interest mainly to specialists in the Supporting 6954

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