Concentration of poliovirus from tap water using positively charged microporous filters.

Microporous filters that are more electropositive than the negatively charged filters currently used for virus concentrations from water by filter adsorption-elution methods were evaluated for poliovirus recovery from tap water. Zeta Plus filters composed of diatomaceous earth-cellulose-"charge-modified" resin mixtures and having a net positive charge of up to pH 5 to 6 efficiently adsorbed poliovirus from tap water at ambient pH levels 7.0 to 7.5 without added multivalent cation salts. The adsorbed virus were eluted with glycine-NaOH, pH 9.5 to 11.5. Electropositive asbestos-cellulose filters efficiently adsorbed poliovirus from tap water without added multivalent cation salts between pH 3.5 and 9.0, and the absorbed viruses could be eluted with 3% beef extract, pH 9, but not with pH 9.5 to 11.5 glycine-NaOH. Under water quality conditions in which poliovirus recoveries from large volumes of water were less than 5% with conventional negatively charged filters and standard methods, recoveries with Zeta Plus filters averaged 64 and 22.5% for one- and two-stage concentration procedures, respectively. Electropositive filters appear to offer distinct advantages over conventional negatively charged filters for concentrating enteric viruses from water, and their behavior tends to confirm the importance of electrostatic forces in virus recovery from water by microporous filter adsorption-elution methods.     

Concentration of poliovirus from tap water using positively charged microporous filters.

Microporous filters that are more electropositive than the negatively charged filters currently used for virus concentrations from water by filter adsorption-elution methods were evaluated for poliovirus recovery from tap water. Zeta Plus filters composed of diatomaceous earth-cellulose-"charge-modified" resin mixtures and having a net positive charge of up to pH 5 to 6 efficiently adsorbed poliovirus from tap water at ambient pH levels 7.0 to 7.5 without added multivalent cation salts. The adsorbed virus were eluted with glycine-NaOH, pH 9.5 to 11.5. Electropositive asbestos-cellulose filters efficiently adsorbed poliovirus from tap water without added multivalent cation salts between pH 3.5 and 9.0, and the absorbed viruses could be eluted with 3% beef extract, pH 9, but not with pH 9.5 to 11.5 glycine-NaOH. Under water quality conditions in which poliovirus recoveries from large volumes of water were less than 5% with conventional negatively charged filters and standard methods, recoveries with Zeta Plus filters averaged 64 and 22.5% for one- and two-stage concentration procedures, respectively. Electropositive filters appear to offer distinct advantages over conventional negatively charged filters for concentrating enteric viruses from water, and their behavior tends to confirm the importance of electrostatic forces in virus recovery from water by microporous filter adsorption-elution methods.     

Concentration of viruses in beef extract by flocculation with ammonium sulfate

Bacteriophages and enteroviruses in water were adsorbed to positively charged filters (Virosorb 1MDS [AMF Cuno, Inc., Meriden, Conn.] or Seitz S [Republic Filters, Milldaler, Conn.]). Adsorbed viruses were eluted by treating the filters with 10% beef extract, pH 9. Organic flocculation of the beef extract at pH 3.5 permitted recovery of more than 40% of the enteroviruses tested but less than 15% of the bacteriophages present. A method was developed that uses salts at pH 7 to flocculate beef extract. Two volumes of saturated ammonium sulfate were added to beef extract, and both enteroviruses and bacteriophages were adsorbed to the flocs that formed. Greater than 70% of the enteroviruses and bacteriophages were recovered by centrifuging the sample and suspending the flocs in a small volume of distilled water.     

Concentration of coliphages from large volumes of water and wastewater.

Membrane filter adsorption-elution technology has been extensively used for the concentration and detection of animal viruses from large volumes of water. This study describes the development of positively charged microporous filters (Zeta Plus) for the concentration of coliphages from large volumes of water and wastewater. Four different coliphages were studied: MS-2, phi X174, T2, and T4. Positively charged microporous filters were found to efficiently adsorb these coliphages from tap water, sewage, and lake water at neutral pH. Adsorbed viruses were eluted with a 1:1 mixture of 8% beef extract and 1 M sodium chloride at pH 9. Using this method, coliphages could be concentrated from 17-liter volumes of tap water with recoveries ranging from 34 to 100%. Coliphages occurring naturally in raw and secondarily treated sewage were recovered with average efficiencies of 56.5 and 55.0%, respectively. This method should be useful in isolation of rare phages, the ecology of phages in natural waters, and the evaluation of water quality.     

Concentration of viruses from water by using cellulose filters modified by in situ precipitation of ferric and aluminum hydroxides.

Untreated cellulose filters adsorbed only small amounts of poliovirus 1, echovirus 5, coxsackievirus B5, or bacteriophage MS2 that were added to tap water or to solutions of imidazole-glycine buffer at pH 5 to 7. Modification of filters by in situ flocculation of ferric and aluminum hydroxides greatly increased the ability of the filters to adsorb viruses. Viruses adsorbed to the modified filters could be recovered by treating the filters with 3% beef extract (pH 9.5). Greater than 60% of the enteroviruses and greater than 55% of the MS2 added to tap water or buffer could be recovered in the beef extract eluate.     

Concentration of viruses from water by using cellulose filters modified by in situ precipitation of ferric and aluminum hydroxides

Untreated cellulose filters adsorbed only small amounts of poliovirus 1, echovirus 5, coxsackievirus B5, or bacteriophage MS2 that were added to tap water or to solutions of imidazole-glycine buffer at pH 5 to 7. Modification of filters by in situ flocculation of ferric and aluminum hydroxides greatly increased the ability of the filters to adsorb viruses. Viruses adsorbed to the modified filters could be recovered by treating the filters with 3% beef extract (pH 9.5). Greater than 60% of the enteroviruses and greater than 55% of the MS2 added to tap water or buffer could be recovered in the beef extract eluate.     

Concentration of viruses from environmental waters using nanoalumina fiber filters

Human viral contamination in drinking and recreational water may persist for extensive periods of time and cause a significant health risk concern. The aim of this study is to evaluate a viral recovery method using a new electropositive charged nanoalumina filter and to compare results with the widely used negatively charged HAWP filter by Millipore Inc. The recovery of infectious recombinant adenovirus type 5 (rAd5) was tested using the Fluorescence-Activated Cell Sorting (FACS) assay, in parallel with viral genomes recovery assay by quantitative PCR (qPCR). The mean infectivity recoveries were 82-91% by nanoalumina filters eluted with 3% beef extract (BE, pH6.0), and 78-90% by HAWP filters eluted with 3% BE (pH 9.0), respectively, from 1 L of environmental samples seeded with 1pfu/mL rAd5. The mean genome recoveries were 16-35% by nanoalumina filters eluted with BE (pH 6.0), and 29-66% by HAWP filters eluted with NaOH (pH 10.8) from different types of water, respectively. Water quality, concentration of viruses, filters, and elution buffers are factors that determine the viral recovery efficiencies. The nanoalumina filters also had higher filtration rates than HAWP filters for large volumes of environmental water samples (up to10 L), thus, have an advantage in concentrating infectious viruses from environments without pre-filtration, adjusting pH or adding multivalent cations.     

Simple method for the concentration of influenza virus from allantoic fluid on microporous filters.

Membrane filter adsorption-elution is an efficient method for concentration and partial purification of several types of viruses from various aqueous solutions. For efficient virus adsorption to negatively charged filters, the sample is adjusted to pH 3.5 and trivalent salts are added before filtration. Since influenza virus is sensitive to extremes in pH, it cannot be concentrated by ordinary filters. Zeta Plus filters, which have a net positive charge of up to 5 or 6, were evaluated for the concentration of influenza virus from infectious allantoic fluids. Influenza virus efficiently adsorbed to Zeta Plus filters at pH 6, and addition of salts was not necessary. Adsorbed virus was eluted in a small volume of 2% bovine serum albumin plus 1 M NaCl at pH 10. By this procedure, viruses in 100 ml of allantoic fluid were concentrated to a final volume of 8 ml, with an average recovery efficiency of 71.0%.     

Comparison of microporous filters for concentration of viruses from wastewater

The 1-MDS Virosorb filter and the 50S and 30S Zeta-plus filters, all with a net positive charge, were compared with the negatively charged Filterite filter for concentration of naturally occurring coliphages and animal viruses from sewage effluent. When Filterite filters were used, the effluent was adjusted to pH 3.5 and AlCl3 was added before filtration to facilitate virus adsorption. No adjustment was required with the positively charged filters. Sets of each filter type were eluted with 3% beef extract (pH 9.5) or eluted with 0.05 M glycine (pH 11.5). A maximum volume of 19 liters could be passed through 142-mm diameter Filterite filters before clogging, whereas only 11, 11, and 15 liters could be passed through the 1-MDS, 50S, and 30S filters, respectively. For equal volumes passed through the filters, coliphage recoveries were 14, 15, 18, and 37% in primary effluent and 40, 97, 50, and 46% in secondary effluent for the Filterite , 1-MDS, 50S, and 30S filters, respectively. No statistically significant difference was observed in the recovery of animal viruses among the filters from secondary effluent, whereas in the Filterite and 50S filters, higher numbers of viruses from primary effluent were recovered than in the 1-MDS and 30S filters in two of three collections. Glycine was found to be a less-efficient eluent than beef extract in the recovery of naturally occurring viruses.     

Comparison of microporous filters for concentration of viruses from wastewater.

The 1-MDS Virosorb filter and the 50S and 30S Zeta-plus filters, all with a net positive charge, were compared with the negatively charged Filterite filter for concentration of naturally occurring coliphages and animal viruses from sewage effluent. When Filterite filters were used, the effluent was adjusted to pH 3.5 and AlCl3 was added before filtration to facilitate virus adsorption. No adjustment was required with the positively charged filters. Sets of each filter type were eluted with 3% beef extract (pH 9.5) or eluted with 0.05 M glycine (pH 11.5). A maximum volume of 19 liters could be passed through 142-mm diameter Filterite filters before clogging, whereas only 11, 11, and 15 liters could be passed through the 1-MDS, 50S, and 30S filters, respectively. For equal volumes passed through the filters, coliphage recoveries were 14, 15, 18, and 37% in primary effluent and 40, 97, 50, and 46% in secondary effluent for the Filterite , 1-MDS, 50S, and 30S filters, respectively. No statistically significant difference was observed in the recovery of animal viruses among the filters from secondary effluent, whereas in the Filterite and 50S filters, higher numbers of viruses from primary effluent were recovered than in the 1-MDS and 30S filters in two of three collections. Glycine was found to be a less-efficient eluent than beef extract in the recovery of naturally occurring viruses.     

Modified membrane-filter procedure for concentration of enteroviruses from tap water.

Enteroviruses added to 114 liters of dechlorinated tap water were recovered in a 16-ml sample by a two-stage concentration procedure in which different types of membrane filters were used in each concentration stage. Viruses in tap water at pH 3.5 were first adsorbed to 10-in. (ca. 25.4-cm) epoxy-fiber glass filters (Filterite). Viruses adsorbed to these filters were eluted with a solution of 0.2 M sodium trichloroacetate buffered at pH 9 with 0.2 M lysine. Viruses in this solution were adsorbed to 47-mm asbestos filters (Seitz) without pH adjustment or other modification of the solution. Viruses were recovered from the Seitz filters with 16 ml of either Casitone or fetal calf serum at pH 9. With these procedures ca. 45% of several types of enteroviruses added to 114 liters of tap water could be recovered in the final 16-ml sample.     

Modified membrane-filter procedure for concentration of enteroviruses from tap water

Enteroviruses added to 114 liters of dechlorinated tap water were recovered in a 16-ml sample by a two-stage concentration procedure in which different types of membrane filters were used in each concentration stage. Viruses in tap water at pH 3.5 were first adsorbed to 10-in. (ca. 25.4-cm) epoxy-fiber glass filters (Filterite). Viruses adsorbed to these filters were eluted with a solution of 0.2 M sodium trichloroacetate buffered at pH 9 with 0.2 M lysine. Viruses in this solution were adsorbed to 47-mm asbestos filters (Seitz) without pH adjustment or other modification of the solution. Viruses were recovered from the Seitz filters with 16 ml of either Casitone or fetal calf serum at pH 9. With these procedures ca. 45% of several types of enteroviruses added to 114 liters of tap water could be recovered in the final 16-ml sample.     

Positively charged filters for virus recovery from wastewater treatment plant effluents.

Positively charged Zeta Plus filters were used to concentrate enteroviruses from 19 liters of effluent from activated sludge units. Neither the addition of salts nor the acidification of the effluent was required for adsorption of viruses to the filters. Viruses adsorbed to the filters were eluted by treating the filters with a solution of 4 M urea buffered at pH 9 with 0.05 M lysine. Eluted viruses were concentrated into final volumes of 1 to 2 ml by using a two-step concentration procedure that employed inorganic and organic flocculation. Approximately 50% of the viruses added to effluents could be recovered in the final sample. The procedure was used to monitor effluents from activated sludge units at two wastewater treatment plants for the presence of enteroviruses.     

Novel approach for modifying microporous filters for virus concentration from water.

Electronegative microporous filters composed of epoxyfiberglass (Filterite) were treated with cationic polymers to enhance their virus-adsorbing properties. This novel and inexpensive approach to microporous filter modification entails soaking filters in an aqueous solution of a cationic polymer such as polyethyleneimine (PEI) for 2 h at room temperature and then allowing the filters to air dry overnight on absorbent paper towels. PEI-treated filters were evaluated for coliphage (MS2, T2, and phi X174) and enterovirus (poliovirus type 1 and coxsackievirus type B5) adsorption from buffer at pH 3.5 to 9.0 and for indigenous coliphages from unchlorinated secondary effluent at ambient pH. Adsorbed viruses were recovered with 3% beef extract (pH 9). Several other cationic polymers were used to modify epoxyfiberglass filters and were evaluated for their ability to concentrate viruses from water. Zeta potentials of disrupted filter material indicated that electronegative epoxyfiberglass filters were made more electropositive when treated with cationic polymers. In general, epoxyfiberglass filters treated with cationic polymers were found to adsorb a greater percentage of coliphages and enteroviruses than were untreated filters.     

Novel approach for modifying microporous filters for virus concentration from water

Electronegative microporous filters composed of epoxyfiberglass (Filterite) were treated with cationic polymers to enhance their virus-adsorbing properties. This novel and inexpensive approach to microporous filter modification entails soaking filters in an aqueous solution of a cationic polymer such as polyethyleneimine (PEI) for 2 h at room temperature and then allowing the filters to air dry overnight on absorbent paper towels. PEI-treated filters were evaluated for coliphage (MS2, T2, and phi X174) and enterovirus (poliovirus type 1 and coxsackievirus type B5) adsorption from buffer at pH 3.5 to 9.0 and for indigenous coliphages from unchlorinated secondary effluent at ambient pH. Adsorbed viruses were recovered with 3% beef extract (pH 9). Several other cationic polymers were used to modify epoxyfiberglass filters and were evaluated for their ability to concentrate viruses from water. Zeta potentials of disrupted filter material indicated that electronegative epoxyfiberglass filters were made more electropositive when treated with cationic polymers. In general, epoxyfiberglass filters treated with cationic polymers were found to adsorb a greater percentage of coliphages and enteroviruses than were untreated filters.     

Evaluation of MK filters for recovery of enteroviruses from tap water.

The MK filter is an electropositively charged filter that can be used to concentrate enteroviruses from large volumes (400 to 1,000 liters) of water. This filter is less expensive than the commonly used 1MDS electropositive filter. In this study, we compared the recovery of poliovirus 1 (PV1) and that of coxsackievirus B3 (CB3) from 378 liters of tap water, using both the MK and the 1MDS filters. Viruses were eluted from the filters with 3% beef extract buffered with 0.05 M glycine (pH 9.5) and reconcentrated via organic flocculation. At high virus inputs (approximately 10(6) PFU), the overall recovery (after elution and reconcentration) of PV1 and CB3 from tap water with the MK filter was less than that achieved with the 1MDS filter (P < 0.05). The recoveries of PV1 from tap water with the MK and 1MDS filters were 73.2% +/- 26% (n = 5 trials) and 90.2% +/- 5.9% (n = 5 trials), respectively. The recoveries of CB3 from tap water with the MK and 1MDS filters were 32.8% +/- 34.5% (n = 4 trials) and 95.8% +/- 12.0% (n = 4 trials), respectively. This study indicated that the MK filter consistently provided lower recovery, with wider variability, of PV1 and CB3 from tap water than the 1MDS filter.     

Poliovirus concentration from tap water with electropositive adsorbent filters.

Simple, reliable, and efficient concentration of poliovirus from tap water was obtained with two types of electropositive filter media, one of which is available in the form of a pleated cartridge filter (Virozorb 1MDS). Virus adsorption from tap water between pH 3.5 and 7.5 was more efficient with electropositive filters than with Filterite filters. Elution of adsorbed viruses was more efficient with beef extract in glycine, pH 9.5, than with glycine-NaOH, pH 11.0. In paired comparative studies, electropositive filters, with adsorption at pH 7.5 and no added polyvalent cation salts, gave less variable virus concentration efficiencies than did Filterite filters with adsorption at pH 3.5 plus added MgCl2. Recovery of poliovirus from 1,000-liter tap water volumes was approximately 30% efficient with both Virozorb 1MDS and Filterite pleated cartridge filters, but the former were much simpler to use. The virus adsorption behavior of these filters appears to be related to their surface charge properties, with more electropositive filters giving more efficient virus adsorption from tap water at higher pH levels.     

Poliovirus concentration from tap water with electropositive adsorbent filters

Simple, reliable, and efficient concentration of poliovirus from tap water was obtained with two types of electropositive filter media, one of which is available in the form of a pleated cartridge filter (Virozorb 1MDS). Virus adsorption from tap water between pH 3.5 and 7.5 was more efficient with electropositive filters than with Filterite filters. Elution of adsorbed viruses was more efficient with beef extract in glycine, pH 9.5, than with glycine-NaOH, pH 11.0. In paired comparative studies, electropositive filters, with adsorption at pH 7.5 and no added polyvalent cation salts, gave less variable virus concentration efficiencies than did Filterite filters with adsorption at pH 3.5 plus added MgCl2. Recovery of poliovirus from 1,000-liter tap water volumes was approximately 30% efficient with both Virozorb 1MDS and Filterite pleated cartridge filters, but the former were much simpler to use. The virus adsorption behavior of these filters appears to be related to their surface charge properties, with more electropositive filters giving more efficient virus adsorption from tap water at higher pH levels.     

Use of bituminous coal as an alternative technique for field concentration of waterborne viruses.

A filter system that sandwiches a bituminous coal preparation between two prefilters was comparable to those presently used to recover human viruses from large volumes of water. This filter was effective over a pH range of 3.0 to 7.0. Poliovirus type 1 recoveries from 100-liter seeded samples of Cincinnati tap water did not vary significantly when compared with those of identical samples processed through Filterite and Millipore filters. In studies with raw domestic sewage, virus recoveries were nearly identical from comparable samples filtered through coal and Millipore disk filters. Thus, the availability of coal makes this filter system an inexpensive analytical tool, especially in developing nations, for virus concentration.