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.     

Concentration of Seeded Simian Rotavirus SA-11 from Potable Waters by Using Talc-Celite Layers and Hydroextraction

There is mounting evidence for the waterborne transmission of diarrhea caused by rotaviruses. As a result, proper techniques are required for their recovery from samples of incriminated water. The combined efficiency of the talc-Celite technique and polyethylene glycol 6000 hydroextraction was, therefore, tested for this purpose, using Simian rotavirus SA-11 and MA-104 cells. Conditioning of the dechlorinated tap water samples was carried out by pH adjustment to 6.0 and the addition of Earle balanced salt solution to a final concentration of 1:100. Passage of a 1-liter volume of such a conditioned sample through a layer containing a mixture of talc (300 mg) and Celite 503 (100 mg) led to the adsorption of nearly 93% of the added SA-11 plaque-forming units. For the recovery of the layer-adsorbed virus, 3% beef extract and 1× tryptose phosphate broth were found to be superior to a variety of other eluents tested. When we tested 100-liter sample volumes, layers containing 1.2 g of talc and 0.4 g of Celite were employed. Virus elution was carried out with 100 ml of tryptose phosphate broth. The eluate was concentrated 10-fold by overnight (4°C) hydroextraction with polyethylene glycol. With a total input virus of 7.0 × 10⁵ and 1.4 × 10² plaque-forming units, the recoveries were about 71 and 59%, respectively.     

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.     

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.     

Elution of viruses from coastal sediments

Enteric viruses were eluted from estuarine sediments by using four organic mixtures; these solutions, with or without various supplements, were compared by determining their abilities to desorb virus from sediments taken from shellfish-harvesting sites. The least effective eluents consisted of glycine buffer, milk preparations, and beef extract paste. When virus type and sediment composition were taken into consideration, higher percentages of virus recovery were achieved with isoelectric casein, powdered beef extract, and nutrient broth mixtures. In addition to the type of eluent used, variations in virus recovery were due to the pH of the eluent, the composition of the sediment, and the type of virus being extracted. No clear distinction between the values of protein and inorganic ion supplements could be made.     

Elution of viruses from coastal sediments.

Enteric viruses were eluted from estuarine sediments by using four organic mixtures; these solutions, with or without various supplements, were compared by determining their abilities to desorb virus from sediments taken from shellfish-harvesting sites. The least effective eluents consisted of glycine buffer, milk preparations, and beef extract paste. When virus type and sediment composition were taken into consideration, higher percentages of virus recovery were achieved with isoelectric casein, powdered beef extract, and nutrient broth mixtures. In addition to the type of eluent used, variations in virus recovery were due to the pH of the eluent, the composition of the sediment, and the type of virus being extracted. No clear distinction between the values of protein and inorganic ion supplements could be made.     

Concentration of poliovirus in water by molecular filtration.

The efficiency of concentrating poliovirus 1 from distilled water samples was determined by using a recirculating-flow molecular filtration system. The most efficient recoveries were achieved against members with a 10,000 nominal molecular weight limit pretreated with flocculated beef extract. This procedure yielded a mean virus recovery of 67%.     

Concentration of poliovirus in water by molecular filtration

The efficiency of concentrating poliovirus 1 from distilled water samples was determined by using a recirculating-flow molecular filtration system. The most efficient recoveries were achieved against members with a 10,000 nominal molecular weight limit pretreated with flocculated beef extract. This procedure yielded a mean virus recovery of 67%.     

Processing and transport of environmental virus samples

Poliovirus-seeded tap water, conditioned with MgCl2 and passed through virus-adsorbing filters, gave better poliovirus recovery than water identically treated but conditioned with AlCl3. Elution of several filter types with beef extract yielded higher recoveries than did elution with glycine. Seeded samples filtered through various filters and stored showed considerable virus loss in 2 days when stored at 4 degrees C, whereas those stored at -70 degrees C gave stable virus recovery up to 4 days. Additionally, the use of antifoam during the elution process reduced foaming and increased virus recovery by 28%.     

Processing and transport of environmental virus samples.

Poliovirus-seeded tap water, conditioned with MgCl2 and passed through virus-adsorbing filters, gave better poliovirus recovery than water identically treated but conditioned with AlCl3. Elution of several filter types with beef extract yielded higher recoveries than did elution with glycine. Seeded samples filtered through various filters and stored showed considerable virus loss in 2 days when stored at 4 degrees C, whereas those stored at -70 degrees C gave stable virus recovery up to 4 days. Additionally, the use of antifoam during the elution process reduced foaming and increased virus recovery by 28%.     

Comparison of four eluents in the recovery of indigenous viruses from raw sludge.

The efficiency of 3% casein hydrolysate (CH), 3% lactalbumin hydrolysate (LH), 3% beef extract (BE), and 10% fetal calf serum (FCS) was compared for the recovery of viruses from raw sludge. CH and LH proved to be inefficient and were eliminated from the study after initial testing. In tests with 20 different samples of raw sludge, beef extract eluted virus in 15 (75%) and FCS revealed virus in 19 (95%) of the samples using BS-C-1 cells. That different eluents were not eluting different viruses from the same sample was shown by the serologic and electron-microscopic examination of 43% (18/42) of the isolates. The identified viruses included members of the entero- (coxsackie B, and polio) and reo-virus groups.     

Comparison of commercial beef extracts and similar materials for recovering viruses from environmental samples

Microbiological- and food-grade beef extracts, protein hydrolytic, enzymatic and autolytic digestion products, and whole protein materials were examined for their potential effectiveness for eluting adsorbed enteroviruses from membrane filters with observed efficiencies ranging from less than 1 to 69%. Concentration of enteroviruses from solutions of these protein and protein-derived products by organic flocculation ranged in efficiency from 2 to 125%. Both elution and concentration were dependent upon virus type, as well as nature, source, and production lot of the material being tested. Determining the efficiency of virus concentration was complicated by virus aggregation and apparent virus inactivation by low pH. Effectiveness of concentrating viruses by organic flocculation from solutions prepared with the various test materials seemed independent of the amount of precipitate produced during the flocculation procedure. Quality assurance tests were proposed by which solutions prepared from beef extracts, whole protein, and protein-derived materials could be evaluated for use in eluting adsorbed viruses from membrane filters and for concentrating viruses by organic flocculation. Food-grade beef extract seemed equal to microbiological-grade beef extract in terms of both virus elution and concentration. Several of the nonbeef extract materials evaluated were as effective as beef extract for virus concentration, but were less effective for virus elution.     

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%.     

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.     

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.     

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%.     

Detection of viruses in drinking water by concentration on magnetic iron oxide.

Discharge of raw domestic wastes containing human enteric viruses into water courses, consumption of untreated water from canals, streams, and shallow wells in villages, and cross-contamination of water in the distribution system because of intermittent water supply in urban areas continue to cause widespread outbreaks of infectious hepatitis in India. To detect a low number of viruses in 50- to 100-liter samples of water, a method was developed with magnetic iron oxide as the virus adsorbent. Poliovirus-seeded dechlorinated tap water, adjusted to pH 3.0 and 0.0005 M AlCl3, was filtered through a 10-g bed of iron oxide sandwiched between two AP20 prefilter pads held in a 142-mm-diameter, stainless-steel holder. Virus was eluted from iron oxide by recirculating three times a 100-ml volume of 3% beef extract, pH 9.0. The eluate was reconcentrated to 5 ml by adjusting to pH 3, adding 1 g of iron oxide, stirring for 30 min, and eluting the readsorbed virus with 5 ml of beef extract, pH 9.0. Virus recovery varied from 60 to 80%. Using the above method, we took a survey of drinking water at three locations in Nagpur during 1976 and found the presence of virus in 7 of 50 samples. The quantity of virus recovered ranged from 1 to 7 plaque-forming units per 30 to 60 liters. Virus was detected in some samples even with residual chlorine. No coliforms were detected in the virus-positive samples.