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.     

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 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.     

A Small Volume Procedure for Viral Concentration from Water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.     

Efficiency of beef extract for the recovery of poliovirus from wastewater effluents.

The efficiency of poliovirus elution from fiber glass cartridge filters (K27), epoxy-fiber glass-asbestos filters (M780), and pleated cartridge filters was assessed by using 3% beef extract (pH 9.0) or 0.1 M glycine (pH 11.5). Poliovirus type I, strain LSc, was seeded into 20- to 25-gallon (ca. 75.6- to 95.6-liter) samples of treated sewage effluent and concentrated by using a filter adsorption-elution technique. Virus elution was accomplished by using either two 600-ml portions of 3% beef extract (pH 9.0), or two 1-liter portions of 0.1 M glycine (pH 11.5). In all experiments, beef extract elution followed by organic flocculation was found to be superior, yielding a mean recovery efficiency of 85%, with recoveries ranging from 68 to 100%. Elution with 0.1 M glycine (pH 11.5) followed by inorganic flocculation resulted in a mean recovery efficiency of 36%. The variable range of recoveries with beef extract could not be significantly improved by varying the type of beef extract or by extending the elution time to 30 min. Second-step reconcentration of 1-liter seeded sewage effluent and renovated wastewater samples indicated that organic flocculation was a more efficient method for virus recovery than inorganic flocculation. Beef extract concentrations of less than 3% were found to be efficient in the recovery of poliovirus from renovated wastewater.     

Efficiency of beef extract for the recovery of poliovirus from wastewater effluents.

The efficiency of poliovirus elution from fiber glass cartridge filters (K27), epoxy-fiber glass-asbestos filters (M780), and pleated cartridge filters was assessed by using 3% beef extract (pH 9.0) or 0.1 M glycine (pH 11.5). Poliovirus type I, strain LSc, was seeded into 20- to 25-gallon (ca. 75.6- to 95.6-liter) samples of treated sewage effluent and concentrated by using a filter adsorption-elution technique. Virus elution was accomplished by using either two 600-ml portions of 3% beef extract (pH 9.0), or two 1-liter portions of 0.1 M glycine (pH 11.5). In all experiments, beef extract elution followed by organic flocculation was found to be superior, yielding a mean recovery efficiency of 85%, with recoveries ranging from 68 to 100%. Elution with 0.1 M glycine (pH 11.5) followed by inorganic flocculation resulted in a mean recovery efficiency of 36%. The variable range of recoveries with beef extract could not be significantly improved by varying the type of beef extract or by extending the elution time to 30 min. Second-step reconcentration of 1-liter seeded sewage effluent and renovated wastewater samples indicated that organic flocculation was a more efficient method for virus recovery than inorganic flocculation. Beef extract concentrations of less than 3% were found to be efficient in the recovery of poliovirus from renovated wastewater.     

Concentration of simian rotavirus SA-11 from tap water by membrane filtration and organic flocculation

Simian rotavirus SA-11 was concentrated from tap water by adsorption to and elution from microporous filters, followed by organic flocculation. Two types of filters were compared for their ability to concentrate the virus. Both Zeta Plus 60S and Cox AA type M-780 filters were efficient for virus adsorption, but the efficiency of virus elution was higher with Zeta Plus than with Cox filters. Optimum conditions for virus recovery from Zeta Plus filters included an input water pH of 6.5 to 7.5 and the use of 3% beef extract (pH 9.0) for elution. Under these conditions, an average of 62 to 100% of the virus was recovered in the concentrate. Organic flocculation was used as a second-step concentration method, with average recoveries of 47 to 69%. When the two methods were used to concentrate small numbers (7 to 75 PFU/liter) of input rotavirus, an average of 75 +/- 40% recovery was achieved. With large volumes of input water, however, recovery was reduced to 16 +/- 7%.     

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.     

Low molecular weight substitutes for beef extract as eluents for poliovirus adsorbed to membrane filters.

Basic solutions of beef extract and casein were able to elute poliovirus adsorbed to four membrane filters with different chemical compositions. Hydrolyzed protein and individual amino acids were able to elute virus adsorbed to certain filters but were unable to elute virus adsorbed to other filters efficiently. A solution of 4 M urea buffered at pH 9 with 0.05 M lysine was able to elute greater than 60% of the virus adsorbed to each of the filters tested. Certain solutions of amino acids were capable of eluting virus adsorbed to one filter but permitted adsorption of virus to another filter with a different chemical composition. Acidic amino acids could interfere with elution of virus from membrane filters. Aromatic compounds with an amino group attached to the ring were good eluents for virus adsorbed to epoxy-fiberglass membrane filters. In contrast, aromatic compounds with other substituents were generally poor eluents.     

Optimum pH levels for eluting enteroviruses from sludge solids with beef extract.

This study demonstrates that elution of enteroviruses from a mixture of primary- and activated-sludge solids with beef extract at pH 9.2 +/- 0.2 may be less efficient than elution with beef extract at pH 7.2 +/- 0.2 and that elution of enteroviruses from extended-aeration-sludge solids with beef extract is at best no more efficient at pH 9.2 +/- 0.2 than at pH 7.2 +/- 0.2. Thus, the common practice of adjusting the pH of beef extract used for eluting enteroviruses from the natural neutral level of the elutant to alkaline levels is unnecessary and probably undesirable.     

Optimum pH levels for eluting enteroviruses from sludge solids with beef extract

This study demonstrates that elution of enteroviruses from a mixture of primary- and activated-sludge solids with beef extract at pH 9.2 +/- 0.2 may be less efficient than elution with beef extract at pH 7.2 +/- 0.2 and that elution of enteroviruses from extended-aeration-sludge solids with beef extract is at best no more efficient at pH 9.2 +/- 0.2 than at pH 7.2 +/- 0.2. Thus, the common practice of adjusting the pH of beef extract used for eluting enteroviruses from the natural neutral level of the elutant to alkaline levels is unnecessary and probably undesirable.     

Concentration of Simian Rotavirus SA-11 from Tap Water by Membrane Filtratiòn and Organic Flocculation

Simian rotavirus SA-11 was concentrated from tap water by adsorption to and elution from microporous filters, followed by organic flocculation. Two types of filters were compared for their ability to concentrate the virus. Both Zeta Plus 60S and Cox AA type M-780 filters were efficient for virus adsorption, but the efficiency of virus elution was higher with Zeta Plus than with Cox filters. Optimum conditions for virus recovery from Zeta Plus filters included an input water pH of 6.5 to 7.5 and the use of 3% beef extract (pH 9.0) for elution. Under these conditions, an average of 62 to 100% of the virus was recovered in the concentrate. Organic flocculation was used as a second-step concentration method, with average recoveries of 47 to 69%. When the two methods were used to concentrate small numbers (7 to 75 PFU/liter) of input rotavirus, an average of 75 ± 40% recovery was achieved. With large volumes of input water, however, recovery was reduced to 16 ± 7%.     

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.     

Assessment of recovery efficiency of beef extract reagents for concentrating viruses from municipal wastewater sludge solids by the organic flocculation procedure

This study was designed to assess the capacity of beef extract reagents to form flocs suitable for virus adsorption. Reagent comparisons resulted in the establishment of a modified organic flocculation procedure to concentrate viruses desorbed from sewage sludge solids with currently available modified powdered beef extracts. The method, based on supplementation with paste beef extract floc, achieved virus recoveries comparable to those obtained with powdered beef extract produced before a 1979 change in the manufacturing process. When primary settled sludge solids originating from mostly domestic waste were eluted with an unsupplemented modified powdered beef extract, high virus recovery efficiency was observed upon concentration by organic flocculation. This appreciable increase might have been due to floc-forming substances that were present in the primary settled sludge. These substances did not appear to be present in settled sludge collected from biologically treated wastes. Apparently, the floc-forming substances had been either removed or substantially altered during biological treatment.     

Assessment of recovery efficiency of beef extract reagents for concentrating viruses from municipal wastewater sludge solids by the organic flocculation procedure.

This study was designed to assess the capacity of beef extract reagents to form flocs suitable for virus adsorption. Reagent comparisons resulted in the establishment of a modified organic flocculation procedure to concentrate viruses desorbed from sewage sludge solids with currently available modified powdered beef extracts. The method, based on supplementation with paste beef extract floc, achieved virus recoveries comparable to those obtained with powdered beef extract produced before a 1979 change in the manufacturing process. When primary settled sludge solids originating from mostly domestic waste were eluted with an unsupplemented modified powdered beef extract, high virus recovery efficiency was observed upon concentration by organic flocculation. This appreciable increase might have been due to floc-forming substances that were present in the primary settled sludge. These substances did not appear to be present in settled sludge collected from biologically treated wastes. Apparently, the floc-forming substances had been either removed or substantially altered during biological treatment.     

Recovery of viruses from water by a modified flocculation procedure for second-step concentration

A reduction in virus recovery efficiencies stemming from a change in the commercial processing of powdered beef extract was reversed by the addition of Celite analytical filter aid. Supplementing beef extract with this silicate is recommended as a modification to the organic flocculation procedure for second-step concentration in monitoring for waterborne viruses. Considerable differences in virus recovery were found among lots of beef extract and Celite preparations; this indicates that the performance of each lot of these substances should be checked before use.