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.     

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.     

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.     

Recovery of Poliovirus from Turbid Estuarine Water on Microporous Filters by the Use of Celite

The application of a new step for recovering poliovirus from moderately to highly turbid estuarine water by the filter virus-adsorption technique was investigated. The experiments were conducted under both (i) laboratory-based conditions (200-ml volumes) where the turbidity was controlled and (ii) simulated field conditions (15- to 100-gal volumes) where the turbidity varied depending upon the hydrology of the raw estuarine water. The new step consisted of adding Celite to the turbid water prior to sampling for virus. In the experiments, the pH of the water was first adjusted to 3.5 and then AlCl(3) was added to 0.0005 M. Celite was added to a concentration of 0.01% and mixed thoroughly. Either an HE Cox M-780 microfilter (Cox Instrument, Div. of Lynch Corp., Detroit, Mich.) or an MF-membrane filter (Millipore Corp., Bedford, Mass.) was used as the virus adsorbent. Virus was eluted from the Celite-filter complex in situ at pH 9 with 5x nutrient broth. In the laboratory-based experiments, when turbidity ranged from 5.0 to 30.0 Jackson turbidity units (JTU), virus recovery ranged from 66 to 89%. In the simulated field experiments, when the turbidity ranged from 8.5 to 80.0 JTU, virus recovery ranged from <1 to 74%, depending upon the multiplicity of virus input and the level of turbidity. The new step greatly improved the filtration-flux of turbid water and significantly reduced the premature clogging problem usually observed with microporous filters.     

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.     

Development of a Simple Method for Concentrating Enteroviruses from Oysters

The development of a simple method for concentrating enteroviruses from oysters is described. In this method viruses in homogenized oyster tissues are efficiently absorbed to oyster solids at pH 5.5 and low salt concentration. After low-speed centrifugation, the supernatant is discarded and viruses are eluted from the sedimented oyster solids by resuspending them in pH 3.5 glycine-buffered saline. The solids are then removed by low-speed centrifugation, and the virus-containing supernatant is filtered through a 0.2-micronm porosity filter to remove bacteria and other small particulates without removing viruses. The virus-containing filtrate is then concentrated to a volume of a few milliliters by ultrafiltration, and the concentrate obtained is inoculated directly into cell cultures for virus assay. When tested with pools of oysters experimentally contaminated with small amounts of different enteroviruses, virus recovery efficiency averaged 63%.     

Influence of salts on virus adsorption to microporous filters

We investigated the direct and indirect effects of mono-, di-, and trivalent salts (NaCl, MgCl(2), and AlCl(3)) on the adsorption of several viruses (MS2, PRD-1, phiX174, 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).     

Preformed magnesium hydroxide precipitate for second-step concentration of enteroviruses from drinking and surface waters

A method is described for the second-step concentration of viruses from large volumes of drinking and surface waters. Seeded viruses present in the first eluate, performed with 50 mM glycine buffer, pH 11.5, were adsorbed on a preformed magnesium hydroxide precipitate. After low-speed centrifugation they were desorbed and adjusted to pH 7 with McIlvaine citrate-phosphate buffer. In these experimental conditions 90% of the viruses present in the 300-mL first eluate were reconcentrated in a final volume of 40 mL. The recovery efficiency was independent of either virus concentration or water quality.     

Improved methods for detecting enteric viruses in oysters.

New and improved methods for concentrating enteroviruses, reoviruses, and adenoviruses from oysters have been developed and evaluated. Viruses are efficiently adsorbed to homogenized oyster meat by adjusting the homogenate to pH 5.0 and a conductivity of less than or equal to 2,000 mg of NaCl per liter. After low-speed centrifugation, the virus-free supernatant is discarded and the viruses are eluted from the sedimented oyster solids with pH 7.5 glycine-NaCl having a conductivity of 8,000 mg of NaCl per liter. The oyster solids are removed by low-speed centrifugation and filtration, and the viruses in the filtered supernatant are concentrated to a small volume by either ultrafiltration or acid precipitation at pH 4.5. The concentrate is treated with antibiotics and inoculated into cell cultures for virus isolation and quantitation. When these methods were tested with oysters experimentally contaminated with polioviruses, reoviruses, and adenoviruses, recovery efficiencies averaged about 46%. With the exception of virus assay and quantitation, these methods are simple and inexpensive enough to be done in typical shellfish microbiology laboratories.     

Improved methods for detecting enteric viruses in oysters.

New and improved methods for concentrating enteroviruses, reoviruses, and adenoviruses from oysters have been developed and evaluated. Viruses are efficiently adsorbed to homogenized oyster meat by adjusting the homogenate to pH 5.0 and a conductivity of less than or equal to 2,000 mg of NaCl per liter. After low-speed centrifugation, the virus-free supernatant is discarded and the viruses are eluted from the sedimented oyster solids with pH 7.5 glycine-NaCl having a conductivity of 8,000 mg of NaCl per liter. The oyster solids are removed by low-speed centrifugation and filtration, and the viruses in the filtered supernatant are concentrated to a small volume by either ultrafiltration or acid precipitation at pH 4.5. The concentrate is treated with antibiotics and inoculated into cell cultures for virus isolation and quantitation. When these methods were tested with oysters experimentally contaminated with polioviruses, reoviruses, and adenoviruses, recovery efficiencies averaged about 46%. With the exception of virus assay and quantitation, these methods are simple and inexpensive enough to be done in typical shellfish microbiology laboratories.     

New method using a positively charged microporous filter and ultrafiltration for concentration of viruses from tap water

The methods used to concentrate enteric viruses from water have remained largely unchanged for nearly 30 years, with the most common technique being the use of 1MDS Virozorb filters followed by organic flocculation for secondary concentration. Recently, a few studies have investigated alternatives; however, many of these methods are impractical for use in the field or share some of the limitations of this traditional method. In the present study, the NanoCeram virus sampler, an electropositive pleated microporous filter composed of microglass filaments coated with nanoalumina fibers, was evaluated. Test viruses were first concentrated by passage of 20 liters of seeded water through the filter (average filter retention efficiency was ≥ 99.8%), and then the viruses were recovered using various salt-based or proteinaceous eluting solutions. A 1.0% sodium polyphosphate solution with 0.05 M glycine was determined to be the most effective. The recovered viruses were then further concentrated using Centricon Plus-70 centrifugal ultrafilters to a final volume of 3.3 (±0.3 [standard deviation]) ml; this volume compares quite favorably to that of previously described methods, such as organic flocculation (~15 to 40 ml). The overall virus recovery efficiencies were 66% for poliovirus 1, 83% for echovirus 1, 77% for coxsackievirus B5, 14% for adenovirus 2, and 56% for MS2 coliphage. In addition, this method appears to be compatible with both cell culture and PCR assays. This new approach for the recovery of viruses from water is therefore a viable alternative to currently used methods when small volumes of final concentrate are an advantage.     

Detection of enteric viruses in treated drinking water.

The occurrence of viruses in conventionally treated drinking water derived from a heavily polluted source was evaluated by collecting and analyzing 38 large-volume (65- to 756-liter) samples of water from a 9 m3/s (205 X 10(6) gallons [776 X 10(6) liters] per day) water treatment plant. Samples of raw, clarified, filtered, and chlorinated finished water were concentrated by using the filter adsorption-elution technique. Of 23 samples of finished water, 19 (83%) contained viruses. None of the nine finished water samples collected during the dry season contained detectable total coliform bacteria. Seven of nine finished water samples collected during the dry season met turbidity, total coliform bacteria, and total residual chlorine standards. Of these, four contained virus. During the dry season the percent removals were 25 to 93% for enteric viruses, 89 to 100% for bacteria, and 81% for turbidity. During the rainy season the percent removals were 0 to 43% for enteric viruses, 80 to 96% for bacteria, and 63% for turbidity. None of the 14 finished water samples collected during the rainy season met turbidity standards, and all contained rotaviruses or enteroviruses.     

Detection of enteric viruses in treated drinking water

The occurrence of viruses in conventionally treated drinking water derived from a heavily polluted source was evaluated by collecting and analyzing 38 large-volume (65- to 756-liter) samples of water from a 9 m3/s (205 X 10(6) gallons [776 X 10(6) liters] per day) water treatment plant. Samples of raw, clarified, filtered, and chlorinated finished water were concentrated by using the filter adsorption-elution technique. Of 23 samples of finished water, 19 (83%) contained viruses. None of the nine finished water samples collected during the dry season contained detectable total coliform bacteria. Seven of nine finished water samples collected during the dry season met turbidity, total coliform bacteria, and total residual chlorine standards. Of these, four contained virus. During the dry season the percent removals were 25 to 93% for enteric viruses, 89 to 100% for bacteria, and 81% for turbidity. During the rainy season the percent removals were 0 to 43% for enteric viruses, 80 to 96% for bacteria, and 63% for turbidity. None of the 14 finished water samples collected during the rainy season met turbidity standards, and all contained rotaviruses or enteroviruses.     

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).     

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%.     

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%.