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.     

Use of modified diatomaceous earth for removal and recovery of viruses in water.

Diatomaceous earth was modified by in situ precipitation of metallic hydroxides. Modification decreased the negative charge on the diatomaceous earth and increased its ability to adsorb viruses in water. Electrostatic interactions were more important than hydrophobic interactions in virus adsorption to modified diatomaceous earth. Filters containing diatomaceous earth modified by in situ precipitation of a combination of ferric chloride and aluminum chloride adsorbed greater than 80% of enteroviruses (poliovirus 1, echovirus 5, and coxsackievirus B5) and coliphage MS2 present in tap water at ambient pH (7.8 to 8.3), even after filtration of 100 liters of tap water. Viruses adsorbed to the filters could be recovered by mixing the modified diatomaceous earth with 3% beef extract plus 1 M NaCl (pH 9).     

Use of modified diatomaceous earth for removal and recovery of viruses in water.

Diatomaceous earth was modified by in situ precipitation of metallic hydroxides. Modification decreased the negative charge on the diatomaceous earth and increased its ability to adsorb viruses in water. Electrostatic interactions were more important than hydrophobic interactions in virus adsorption to modified diatomaceous earth. Filters containing diatomaceous earth modified by in situ precipitation of a combination of ferric chloride and aluminum chloride adsorbed greater than 80% of enteroviruses (poliovirus 1, echovirus 5, and coxsackievirus B5) and coliphage MS2 present in tap water at ambient pH (7.8 to 8.3), even after filtration of 100 liters of tap water. Viruses adsorbed to the filters could be recovered by mixing the modified diatomaceous earth with 3% beef extract plus 1 M NaCl (pH 9).     

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 large volumes of tap water using pleated membrane filters.

A method is described for the efficient concentration of viruses from large volumes of tap water in relatively short time periods. Virus in acidified tap water in the presence of aluminum chloride is adsorbed to a 10-inch (ca. 25.4 cm) fiberglass depth cartridge and a 10-inch pleated epoxy-fiberglass filter in series at flow rates of up to 37.8 liters/min (10 gallons/min). This filter series is capable of efficiently adsorbing virus from greater than 19,000 liters (5,000 gallons) of treated tap water. Adsorbed viruses are eluted from the filters with glycine buffer (pH 10.5) and the eluate is reconcentrated using an aluminum flocculation process. Viruses are eluted from the aluminum floc with glycine buffer (pH 11.5). Using this procedure, viruses in 1,900 liters (500 gallons) of tap water can be concentrated 100,000-fold in 3 h with an average recovery of 40 to 50%.     

Concentration of viruses from large volumes of tap water using pleated membrane filters.

A method is described for the efficient concentration of viruses from large volumes of tap water in relatively short time periods. Virus in acidified tap water in the presence of aluminum chloride is adsorbed to a 10-inch (ca. 25.4 cm) fiberglass depth cartridge and a 10-inch pleated epoxy-fiberglass filter in series at flow rates of up to 37.8 liters/min (10 gallons/min). This filter series is capable of efficiently adsorbing virus from greater than 19,000 liters (5,000 gallons) of treated tap water. Adsorbed viruses are eluted from the filters with glycine buffer (pH 10.5) and the eluate is reconcentrated using an aluminum flocculation process. Viruses are eluted from the aluminum floc with glycine buffer (pH 11.5). Using this procedure, viruses in 1,900 liters (500 gallons) of tap water can be concentrated 100,000-fold in 3 h with an average recovery of 40 to 50%.     

Concentration of enteroviruses from estuarine water.

Pleated cartridge filters readily adsorb viruses in estuarine water at low pH containing aluminum chloride. Adsorbed viruses are efficiently recovered by treating filters with glycine buffer at high pH. By using these procedures, it was possible to recover approximately 70% of the poliovirus added to 400 liters of estuarine water in 3 liters of filter eluate. Reconcentration of virus in the filter eluate in small volumes that are convenient for viral assays was more difficult. Reconcentration methods described previously for eluates from filters that process tap water or treated wastewater were inadequate when applied to eluates from filters used to process estuarine water containing large amounts of organic compounds. Two methods were found to permit efficient concentration of virus in filter eluates in small volumes. In both methods, virus in 3 liters of filter eluate was adsorbed to aluminum hydroxide flocs and then recovered in approximately 150 ml of buffered fetal calf serum. Additional reductions in volume were achieved by ultrafiltration or hydroextraction. By using these procedures 60 to 80% of the virus in 3 liters of filter eluate could be recovered in a final volume of 10 to 40 ml.     

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 enteroviruses from estuarine water.

Pleated cartridge filters readily adsorb viruses in estuarine water at low pH containing aluminum chloride. Adsorbed viruses are efficiently recovered by treating filters with glycine buffer at high pH. By using these procedures, it was possible to recover approximately 70% of the poliovirus added to 400 liters of estuarine water in 3 liters of filter eluate. Reconcentration of virus in the filter eluate in small volumes that are convenient for viral assays was more difficult. Reconcentration methods described previously for eluates from filters that process tap water or treated wastewater were inadequate when applied to eluates from filters used to process estuarine water containing large amounts of organic compounds. Two methods were found to permit efficient concentration of virus in filter eluates in small volumes. In both methods, virus in 3 liters of filter eluate was adsorbed to aluminum hydroxide flocs and then recovered in approximately 150 ml of buffered fetal calf serum. Additional reductions in volume were achieved by ultrafiltration or hydroextraction. By using these procedures 60 to 80% of the virus in 3 liters of filter eluate could be recovered in a final volume of 10 to 40 ml.     

Detection of noroviruses in tap water in Japan by means of a new method for concentrating enteric viruses in large volumes of freshwater

A virus concentration method using a cation-coated filter was developed for large-volume freshwater applications. Poliovirus type 1 (LSc 2ab Sabin strain) inoculated into 40 ml of MilliQ (ultrapure) water was adsorbed effectively to a negatively charged filter (Millipore HA, 0.45- micro m pore size) coated with aluminum ions, 99% (range, 81 to 114%) of which were recovered by elution with 1.0 mM NaOH (pH 10.8) following an acid rinse with 0.5 mM H(2)SO(4) (pH 3.0). More than 80% poliovirus recovery yields were obtained from 500-ml, 1,000-ml, and 10-liter MilliQ water samples and from tap water samples. This method, followed by TaqMan PCR detection, was applied to determine the presence of noroviruses in tap water in Tokyo, Japan. In a 14-month survey, 4 (4.1%) and 7 (7.1%) of 98 tap water samples (100 to 532 liters) contained a detectable amount of noroviruses of genotype 1 and genotype 2, respectively. This method was proved to be useful for surveying the occurrence of enteric viruses, including noroviruses, in large volumes of freshwater.     

Detection of Noroviruses in Tap Water in Japan by Means of a New Method for Concentrating Enteric Viruses in Large Volumes of Freshwater

A virus concentration method using a cation-coated filter was developed for large-volume freshwater applications. Poliovirus type 1 (LSc 2ab Sabin strain) inoculated into 40 ml of MilliQ (ultrapure) water was adsorbed effectively to a negatively charged filter (Millipore HA, 0.45-μm pore size) coated with aluminum ions, 99% (range, 81 to 114%) of which were recovered by elution with 1.0 mM NaOH (pH 10.8) following an acid rinse with 0.5 mM H₂SO₄ (pH 3.0). More than 80% poliovirus recovery yields were obtained from 500-ml, 1,000-ml, and 10-liter MilliQ water samples and from tap water samples. This method, followed by TaqMan PCR detection, was applied to determine the presence of noroviruses in tap water in Tokyo, Japan. In a 14-month survey, 4 (4.1%) and 7 (7.1%) of 98 tap water samples (100 to 532 liters) contained a detectable amount of noroviruses of genotype 1 and genotype 2, respectively. This method was proved to be useful for surveying the occurrence of enteric viruses, including noroviruses, in large volumes of freshwater.     

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.     

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.     

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.     

Survival of enteric viruses adsorbed on glass microfibers during postal transport

The survival of enteric viruses (poliovirus type 1, Mahoney strain, and indigenous viruses of waste waters) has been studied after adsorption of the viruses (pH 3.5) on glass microfiber filters. After postal transport, the presence of the viruses was checked on the filters being soaked in a 3% beef extract solution (pH 7.5) either frozen or without protection against heat. Viruses were recovered at a rate of 59 to 65%. For qualitative studies, postal shipment of samples adsorbed on fiberglass may allow extension of a control system for enteroviruses in water.     

Concentration of poliovirus from tap water onto membrane filters with aluminum chloride at ambient pH levels.

A method is described for the concentration of an enterovirus from large volumes of tap water by addition of small amounts of aluminum chloride to enhance virus removal by membrane filters. Tap water treated with 2 X 10(-5) M aluminum chloride showed a slight decrease in pH (less than 0.5), a slight increase in turbidity, and enhanced removal of poliovirus by membrane filters. Virus was quantitatively recovered by treating the filters with a basic buffer, and this eluate was reconcentrated to a small volume by adsorption to aluminum hydroxide flocs. Using these procedures, virus from 1,000 liters of water was reduced to a final eluate of 20 to 80 ml with a mean recovery of 70%.