Influence of Salts on Virus Adsorption to Microporous Filters

We investigated the direct and indirect effects of mono-, di-, and trivalent salts (NaCl, MgCl₂, and AlCl₃) on the adsorption of several viruses (MS2, PRD-1, X174, and poliovirus 1) to microporous filters at different pH values. The filters studied included Millipore HA (nitrocellulose), Filterite (fiberglass), Whatman (cellulose), and 1MDS (charged-modified fiber) filters. Each of these filters except the Whatman cellulose filters has been used in virus removal and recovery procedures. The direct effects of added salts were considered to be the effects associated with the presence of the soluble salts. The indirect effects of the added salts were considered to be (i) changes in the pH values of solutions and (ii) the formation of insoluble precipitates that could adsorb viruses and be removed by filtration. When direct effects alone were considered, the salts used in this study promoted virus adsorption, interfered with virus adsorption, or had little or no effect on virus adsorption, depending on the filter, the virus, and the salt. Although we were able to confirm previous reports that the addition of aluminum chloride to water enhances virus adsorption to microporous filters, we found that the enhanced adsorption was associated with indirect effects rather than direct effects. The increase in viral adsorption observed when aluminum chloride was added to water was related to the decrease in the pH of the water. Similar results could be obtained by adding HCl. The increased adsorption of viruses in water at pH 7 following addition of aluminum chloride was probably due to flocculation of aluminum, since removal of flocs by filtration greatly reduced the enhancement observed. The only direct effect of aluminum chloride on virus adsorption that we observed was interference with adsorption to microporous filters. Under conditions under which hydrophobic interactions were minimal, aluminum chloride interfered with virus adsorption to Millipore, Filterite, and 1MDS filters. In most cases, less than 10% of the viruses adsorbed to filters in the presence of a multivalent salt and a compound that interfered with hydrophobic interactions (0.1% Tween 80 or 4 M urea).     

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.     

Efficiency of several micro-fiber glass filters for recovery of poliovirus from tape water.

Micro-fiber glass filters from Gelman, Filterite, Johns-Manville, and Whatman were compared with Millipore membrane filters on the basis of their virus adsorbancy, flow rate, clogging resistance, and virus concentration efficiency by using tap water at 2 nephelometric turbidity units. As virus adsorbants the Johns-Manville D39, Filterite 0.25-micron, Filterite 0.45-micron, and Millipore 0.45-micron filters were the most efficient, retaining more than 99% of the added virus in water at pH 3.5 and 0.0005 M aluminum chloride. The Johns-Manville D79 and D49 filters retained 92 and 96% of the virus, respectively, whereas the Whatman GF-D, Whatman GF-F, Gelman A-E, and Millipore AP-20 filters retained only 28, 78, 56, and 34% of the virus, respectively. The best flow rate and clogging resistance were obtained with the Johns-Manville D79 filter or with this filter acting as a prefilter to the Johns-Manville D49, Johns-Manville D39, or Filterite 0.45-micron filter. Finally, poliovirus experimentally seeded in 20 liters of tape water was recovered from Johns-Manville D79-Johns-Manville D39 or Johns-Manville D79-Filterite 0.45 micron 142-mm filter combinations was a efficiencies of 86 and 85%, respectively.     

Effects of bentonite clay solids on poliovirus concentration from water by microporous filter methods.

To determine whether suspended solids interfere with enteric virus recovery from water by microporous filter methods, the effects of bentonite clay solids at a concentration of 10 nephelometric turbidity units on the recovery of poliovirus type 1 from seeded, activated carbon-treated, filtered tap water were studied. Volumes (500 ml) of virus-laden water at pH 5.5 or 7.5, with and without 50 mM MgCl2, were filtered through 47-mm-diameter, electropositive (Virosorb 1MDS) and electronegative (Filterite) filters that had been pretreated with Tween 80 to minimize direct virus adsorption to filter surfaces. Bentonite solids enhanced virus retention on both types of filters, even under conditions in which viruses were not solids associated. However, bentonite solids also interfered with elution of retained viruses when eluting with 0.3% beef extract-50 mM glycine (pH 9.5). Under some conditions, overall virus recoveries were lower from water with bentonite solids than from solids-free control water. The results of this study indicate that clay turbidity can interfere somewhat with virus recovery by current microporous filter methods.     

Effects of bentonite clay solids on poliovirus concentration from water by microporous filter methods

To determine whether suspended solids interfere with enteric virus recovery from water by microporous filter methods, the effects of bentonite clay solids at a concentration of 10 nephelometric turbidity units on the recovery of poliovirus type 1 from seeded, activated carbon-treated, filtered tap water were studied. Volumes (500 ml) of virus-laden water at pH 5.5 or 7.5, with and without 50 mM MgCl2, were filtered through 47-mm-diameter, electropositive (Virosorb 1MDS) and electronegative (Filterite) filters that had been pretreated with Tween 80 to minimize direct virus adsorption to filter surfaces. Bentonite solids enhanced virus retention on both types of filters, even under conditions in which viruses were not solids associated. However, bentonite solids also interfered with elution of retained viruses when eluting with 0.3% beef extract-50 mM glycine (pH 9.5). Under some conditions, overall virus recoveries were lower from water with bentonite solids than from solids-free control water. The results of this study indicate that clay turbidity can interfere somewhat with virus recovery by current microporous filter methods.     

Influence of water quality on enteric virus concentration by microporous filter methods.

Four enteric viruses, poliovirus type 1, echovirus type 1, reovirus type 3, and simian adenovirus SV-11, were concentrated from seeded 1.3-liter volumes of raw, finished, and granular activated carbon-treated waters by adsorption to 47-mm-diameter (17 cm2), electropositive ( Virosorb 1MDS ) filters at pH 7.5 or electronegative ( Filterite ) filters at pH 3.5 with and without 5 mM added MgCl2, followed by elution with 0.3% beef extract in 50 mM glycine at pH 9.5. Removal of particulates from raw and finished waters by 0.2-micron prefiltration before virus addition and pH adjustment had little effect on virus concentration efficiencies. Soluble organic compounds reduced virus adsorption efficiencies from both raw and finished waters compared with granular activated carbon-treated water, but the extent of interference varied with virus type and adsorption conditions. For electropositive 1MDS filters, organic interference was similar with all virus types. For Filterite filters, organic interference was evident with poliovirus and echovirus, but could be overcome by adding MgCl2. Reovirus and SV-11 were not adversely affected by organics during adsorption to Filterite filters. Elution of reovirus and adenovirus was inefficient compared with that of poliovirus and echovirus. None of the three adsorption schemes ( 1MDS at pH 7.5 and Filterite with and without 5 mM MgCl2 at pH 3.5) could be judged superior for all viruses and water types tested.     

Influence of water quality on enteric virus concentration by microporous filter methods

Four enteric viruses, poliovirus type 1, echovirus type 1, reovirus type 3, and simian adenovirus SV-11, were concentrated from seeded 1.3-liter volumes of raw, finished, and granular activated carbon-treated waters by adsorption to 47-mm-diameter (17 cm2), electropositive ( Virosorb 1MDS ) filters at pH 7.5 or electronegative ( Filterite ) filters at pH 3.5 with and without 5 mM added MgCl2, followed by elution with 0.3% beef extract in 50 mM glycine at pH 9.5. Removal of particulates from raw and finished waters by 0.2-micron prefiltration before virus addition and pH adjustment had little effect on virus concentration efficiencies. Soluble organic compounds reduced virus adsorption efficiencies from both raw and finished waters compared with granular activated carbon-treated water, but the extent of interference varied with virus type and adsorption conditions. For electropositive 1MDS filters, organic interference was similar with all virus types. For Filterite filters, organic interference was evident with poliovirus and echovirus, but could be overcome by adding MgCl2. Reovirus and SV-11 were not adversely affected by organics during adsorption to Filterite filters. Elution of reovirus and adenovirus was inefficient compared with that of poliovirus and echovirus. None of the three adsorption schemes ( 1MDS at pH 7.5 and Filterite with and without 5 mM MgCl2 at pH 3.5) could be judged superior for all viruses and water types tested.     

Effects of humic and fulvic acids on poliovirus concentration from water by microporous filtration

Because naturally occurring organic matter is thought to interfere with virus adsorption to microporous filters, humic and fulvic acids isolated from a highly colored, soft surface water were used as model organics in studies on poliovirus adsorption to and recovery from electropositive Virosorb 1MDS and electronegative Filterite filters. Solutions of activated carbon-treated tap water containing 3, 10, and 30-mg/liter concentrations of humic or fulvic acid were seeded with known amounts of poliovirus and processed with Virosorb 1MDS filters at pH 7.5 or Filterite filters at pH 3.5 (with and without 5 mM MgCl2). Organic acids caused appreciable reductions in virus adsorption and recovery efficiencies with both types of filter. Fulvic acid caused greater reductions in poliovirus recovery with Virosorb 1MDS filters than with Filterite filters. Fulvic acid interference with poliovirus recovery by Filterite filters was overcome by the presence of 5 mM MgCl2. Although humic acid reduced poliovirus recoveries by both types of filter, its greatest effect was on virus elution and recovery from Filterite filters. Single-particle analyses demonstrated MgCl2 enhancement of poliovirus association with both organic acids at pH 3.5. The mechanisms by which each organic acid reduced virus adsorption and recovery appeared to be different for each type of filter.     

Effects of humic and fulvic acids on poliovirus concentration from water by microporous filtration.

Because naturally occurring organic matter is thought to interfere with virus adsorption to microporous filters, humic and fulvic acids isolated from a highly colored, soft surface water were used as model organics in studies on poliovirus adsorption to and recovery from electropositive Virosorb 1MDS and electronegative Filterite filters. Solutions of activated carbon-treated tap water containing 3, 10, and 30-mg/liter concentrations of humic or fulvic acid were seeded with known amounts of poliovirus and processed with Virosorb 1MDS filters at pH 7.5 or Filterite filters at pH 3.5 (with and without 5 mM MgCl2). Organic acids caused appreciable reductions in virus adsorption and recovery efficiencies with both types of filter. Fulvic acid caused greater reductions in poliovirus recovery with Virosorb 1MDS filters than with Filterite filters. Fulvic acid interference with poliovirus recovery by Filterite filters was overcome by the presence of 5 mM MgCl2. Although humic acid reduced poliovirus recoveries by both types of filter, its greatest effect was on virus elution and recovery from Filterite filters. Single-particle analyses demonstrated MgCl2 enhancement of poliovirus association with both organic acids at pH 3.5. The mechanisms by which each organic acid reduced virus adsorption and recovery appeared to be different for each type of filter.     

Evaluation of methods for concentrating hepatitis A virus from drinking water.

By using recently developed cultivation and assay systems, currently available methods for concentrating enteric viruses from drinking water by adsorption to and subsequent elution from microporous filters followed by organic flocculation were evaluated for their ability to recover hepatitis A virus (HAV). Cell culture-adapted HAV (strain HM-175) in seeded tapwater was efficiently adsorbed by both electronegative (Filterite) and electropositive (Virosorb 1MDS) filters at pH and ionic conditions previously used for other enteric viruses. Adsorbed HAV was efficiently eluted from these filters by beef extract eluents at pH 9.5. Eluted HAV was further concentrated efficiently by acid precipitation (organic flocculation) of eluents containing beef extract made from powdered, but not paste, sources. By using optimum adsorption conditions for each type of filter, HAV was concentrated greater than 100-fold from samples of seeded tapwater, with about 50% recovery of the initial infectious virus added to the samples. The ability to recover and quantify HAV in contaminated drinking water with currently available methods should prove useful in further studies to determine the role of drinking water in HAV transmission.     

Evaluation of methods for concentrating hepatitis A virus from drinking water

By using recently developed cultivation and assay systems, currently available methods for concentrating enteric viruses from drinking water by adsorption to and subsequent elution from microporous filters followed by organic flocculation were evaluated for their ability to recover hepatitis A virus (HAV). Cell culture-adapted HAV (strain HM-175) in seeded tapwater was efficiently adsorbed by both electronegative (Filterite) and electropositive (Virosorb 1MDS) filters at pH and ionic conditions previously used for other enteric viruses. Adsorbed HAV was efficiently eluted from these filters by beef extract eluents at pH 9.5. Eluted HAV was further concentrated efficiently by acid precipitation (organic flocculation) of eluents containing beef extract made from powdered, but not paste, sources. By using optimum adsorption conditions for each type of filter, HAV was concentrated greater than 100-fold from samples of seeded tapwater, with about 50% recovery of the initial infectious virus added to the samples. The ability to recover and quantify HAV in contaminated drinking water with currently available methods should prove useful in further studies to determine the role of drinking water in HAV transmission.     

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.     

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.     

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.     

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.     

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.