Recovery of viruses from vegetable surfaces

The efficiency of a system developed for the recovery of viruses contaminating large quantities of vegetables was investigated in the laboratory and tested in the field. Viruses seeded onto a number of leafy vegetables in the laboratory were eluted with a phosphate-buffered saline solution (pH 9.0). The eluate was clarified by glass wool filtration, and any viruses present were concentrated by adsorption to a Filterite pleated cartridge filter, eluted with 3% beef extract (pH 9.0), and further concentrated by organic flocculation. At least 24 liters of vegetable eluate could be concentrated to 70 to 80 ml, equivalent to a greater than 99.5% reduction in volume. With this system, poliovirus was recovered with a mean efficiency of 58% for all vegetables tested. Adenovirus was recovered from lettuce with a slightly lower mean efficiency (55%). Poliovirus was recovered from large quantities of cabbage for up to 5 days in the field after spray irrigation of relatively low levels of virus, even when heavy rain fell before sampling.     

Concentration of enteroviruses from large volumes of tap water, treated sewage, and seawater.

Methods are described for the efficient concentration of an enterovirus from large volumes of tap water, sewage, and seawater. Virus in acidified water (pH 3.5) in the presence of aluminum chloride was adsorbed to a 10-inch (ca. 25.4 cm) fiberglass depth cartridge and a 10-inch pleated epoxy-fiberglass filter in a series at flow rates of up to 37.8 liters (10 gallons) per min. Adsorbed viruses were eluted from the filters with glycine buffer (pH 10.5 to 11.5), and the eluate was reconcentrated by using a combination of aluminum flocculation followed by hydroextraction. With this procedure, poliovirus in large volumes of tap water, seawater, and sewage could be concentrated with an average efficiency of 52, 53, and 50%, respectively. It was demonstrated that this method is capable of detecting surface solid-associated viruses originating from sewage treatment plants. No difference in virus recovery between laboratory batch studies and a set-up with acid-salt injection was found. This unified scheme for the concentration of viruses has many advantages over previously described systems. These include: high operating flow rates, low weight and small size, effectiveness with a variety of waters with widely varying qualities, and filters with a high resistance to clogging.     

Concentration of enteroviruses from large volumes of tap water, treated sewage, and seawater.

Methods are described for the efficient concentration of an enterovirus from large volumes of tap water, sewage, and seawater. Virus in acidified water (pH 3.5) in the presence of aluminum chloride was adsorbed to a 10-inch (ca. 25.4 cm) fiberglass depth cartridge and a 10-inch pleated epoxy-fiberglass filter in a series at flow rates of up to 37.8 liters (10 gallons) per min. Adsorbed viruses were eluted from the filters with glycine buffer (pH 10.5 to 11.5), and the eluate was reconcentrated by using a combination of aluminum flocculation followed by hydroextraction. With this procedure, poliovirus in large volumes of tap water, seawater, and sewage could be concentrated with an average efficiency of 52, 53, and 50%, respectively. It was demonstrated that this method is capable of detecting surface solid-associated viruses originating from sewage treatment plants. No difference in virus recovery between laboratory batch studies and a set-up with acid-salt injection was found. This unified scheme for the concentration of viruses has many advantages over previously described systems. These include: high operating flow rates, low weight and small size, effectiveness with a variety of waters with widely varying qualities, and filters with a high resistance to clogging.     

Concentration of enteroviruses from large volumes of water

An improved method for concentrating viruses from large volumes of clean waters is described. It was found that, by acidification, viruses in large volumes of water could be efficiently adsorbed to epoxy-fiber-glass and nitrocellulose filters in the absence of exogenously added salts. Based upon this finding, a modified version of our previously described virus concentration system was developed for virus monitoring of clean waters. In this procedure the water being tested is acidified by injection of N HCl prior to passage through a virus adsorber consisting of a fiber-glass cartridge depth filter and an epoxy-fiber-glass membrane filter in series. The adsorbed viruses are then eluted with a 1-liter volume of pH 11.5 eluent and reconcentrated by adsorption to and elution from a small epoxy-fiber-glass filter series. With this method small quantities of poliovirus in 100-gallon (378.5-liter) volumes of tapwater were concentrated nearly 40,000-fold with an average virus recovery efficiency of 77%.     

Concentration of enteroviruses from large volumes of turbid estuary water.

A method is described for the efficient concentration of viruses from large volumes of highly turbid estuary water. Virus in acidified seawater in the presence of aluminum chloride is adsorbed to a 10-in. (about 25.4 cm) fibreglass depth cartridge and 2- and 0.65-micron epoxy-fibreglass filters in series. This filter series is capable of efficiently adsorbing enteroviruses from 50 U.S. gallons (about 190) of estuary water of varying salinity and turbidity. Adsorbed viruses were eluted from the filters with glycine buffer (pH 11.5) and the eluate reconcentrated by using a precipitate formed by the addition of ferric chloride. Viruses were eluted from this precipitate with fetal calf serum. Using this procedure, four different enteroviruses in 50 gallons (about 190) of estuary water were concentrated 9 000- to 12 000-fold with an overall efficiency of 41%.     

Development of a quantitative method for the detection of enteroviruses in soil.

A method is described for efficiently concentrating enteroviruses from soil. Viruses were eluted from soil by mechanical agitation in high pH glycine buffer containing ethylenediaminetetraacetic acid. The eluted viruses were concentrated on a floc that formed de novo upon adjustment of the soil eluate to 0.06 M aluminum chloride and pH 3.5. Viruses not pelleted with the floc were concentrated by adsorption to and elution from membrane filters. This method yielded an average efficiency of 66% recovery from loamy sand soil for four enteroviruses. Virus recovery from soil was consistently high, with samples ranging in size from 25 to 500 g. The method was used successfully to isolate naturally occurring viruses from soil beneath a wastewater land treatment site. Recovery of enteroviruses by this method form different types of soil was dependent on percentage of clay, surface area, and cation exchange capacity. Recovery was not dependent on soil saturation pH or on percentage of organic matter. This method should prove useful for studying enterovirus migration and survival during the land application of domestic sewage.     

Development of a quantitative method for the detection of enteroviruses in soil.

A method is described for efficiently concentrating enteroviruses from soil. Viruses were eluted from soil by mechanical agitation in high pH glycine buffer containing ethylenediaminetetraacetic acid. The eluted viruses were concentrated on a floc that formed de novo upon adjustment of the soil eluate to 0.06 M aluminum chloride and pH 3.5. Viruses not pelleted with the floc were concentrated by adsorption to and elution from membrane filters. This method yielded an average efficiency of 66% recovery from loamy sand soil for four enteroviruses. Virus recovery from soil was consistently high, with samples ranging in size from 25 to 500 g. The method was used successfully to isolate naturally occurring viruses from soil beneath a wastewater land treatment site. Recovery of enteroviruses by this method form different types of soil was dependent on percentage of clay, surface area, and cation exchange capacity. Recovery was not dependent on soil saturation pH or on percentage of organic matter. This method should prove useful for studying enterovirus migration and survival during the land application of domestic sewage.     

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 viruses from environmental waters using nanoalumina fiber filters

Human viral contamination in drinking and recreational water may persist for extensive periods of time and cause a significant health risk concern. The aim of this study is to evaluate a viral recovery method using a new electropositive charged nanoalumina filter and to compare results with the widely used negatively charged HAWP filter by Millipore Inc. The recovery of infectious recombinant adenovirus type 5 (rAd5) was tested using the Fluorescence-Activated Cell Sorting (FACS) assay, in parallel with viral genomes recovery assay by quantitative PCR (qPCR). The mean infectivity recoveries were 82-91% by nanoalumina filters eluted with 3% beef extract (BE, pH6.0), and 78-90% by HAWP filters eluted with 3% BE (pH 9.0), respectively, from 1 L of environmental samples seeded with 1pfu/mL rAd5. The mean genome recoveries were 16-35% by nanoalumina filters eluted with BE (pH 6.0), and 29-66% by HAWP filters eluted with NaOH (pH 10.8) from different types of water, respectively. Water quality, concentration of viruses, filters, and elution buffers are factors that determine the viral recovery efficiencies. The nanoalumina filters also had higher filtration rates than HAWP filters for large volumes of environmental water samples (up to10 L), thus, have an advantage in concentrating infectious viruses from environments without pre-filtration, adjusting pH or adding multivalent cations.     

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.     

An evaluation of elution techniques in the study of immune complex glomerulonephritis.

Antigen and antibody from glomerular immune complex deposits in rabbits with experimental bovine serum albumin-(BSA) induced chronic serum sickness (CSS) were quantitated in elutes from kidneys in which a portion of the antigen and antibody had been radiolabeled. The largest quantities of 125I BSA eluted with 1 M roprionic acid at pH 2.7 (86%) and 0.1 M borate buffer at pH 11.25 (80%). However, these buffers yielded less functional anti-BSA antibody than 0.02 M citrate buffer at pH 3.2 (344 mug/g kidney). Citrate buffer-eluted anti-BSA antibody was reactive in immunodiffusion, immunofluorescence, and radiolabeled BSA binding test systems, but complement fixation was impaired relative to chaotropic ion-eluted antibody. It was found that up to 75% of the eluted antibody was lost to further study by recombination with eluted BSA. This could be prevented by fractionation of the dissociated eluate before neutralization. IgG fractionated eluates were successfully fluorescein conjugated or radiolabeled for use as reagents. Elution of cryostat sections of CSS kidney was also studied; BSA, IgG, and complement (C3) eluted in parallel, and sub-microgram quantities of anti-BSA antibody were recovered.     

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 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.     

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.     

Second-step concentration of viruses in drinking and surface waters using polyethylene glycol hydroextraction.

In our laboratory, virus adsorbed to talc--Celite layers is eluted with 100 mL of 10% fetal calf serum (FCS) in normal saline (pH 9.0). A further 10-fold reduction in the volume of the eluate was necessary before its inoculation into cell cultures. A 100-mL volume of an experimentally contaminated sample was placed in a dialysis sac and hydroextracted overnight (4 degrees C) with polyethylene glycol (PEG) 6000. The viscous material remaining in the sac was resuspended in 10 mL of Earle's balanced salt solution. After membrane filtration (0.2 micron), the concentrate was plaque assayed in BS-C-1 cells. Using this technique, recoveries of five laboratory-adapted enteric viruses (polio 1, echo 6, coxsackie B5, coxackie A9, and reo 3) and four freshly isolated enteric virus strains (polio 1, echo 1, coxsackie B3, and reo) ranged from 87 to 97%. In comparative tests, PEG hydroextraction was simpler and superior to organic flocculation.     

Development of a quantitative method for detecting enteroviruses in estuarine sediments.

Several investigators have reported on the detection of enteric viruses in marine sediments, but none determined the efficiency of their methods and only limited volumes of sediment were sampled. The purpose of this investigation was to develop a quantitative method for detecting enteroviruses in marine sediments so that their relative proportion to viruses freely suspended in estuarine water could be more accurately determined. Poliovirus was found to adsorb readily to natural marine sediments collected along the Texas Gulf coast. A number of substances were evaluated for their ability to elute adsorbed viruses. A solution of 10% fetal calf serum adjusted to pH 10.5 and 0.05M ethylenediaminetetraacetate (pH 11.0) were found to be the best eluents. Using ethylenediaminetetraacetate as an eluent, it was possible to elute virus from large volumes of sediment and reconcentrate the sediment eluate into an economically assayable volume (30 to 50 ml). Poliovirus could be recovered from the sediment with an overall efficiency of 50%. This method was found to be satisfactory for the recovery of naturally occurring animal viruses in estuarine sediments from the upper Texas Gulf coast.     

Development of a quantitative method for detecting enteroviruses in estuarine sediments.

Several investigators have reported on the detection of enteric viruses in marine sediments, but none determined the efficiency of their methods and only limited volumes of sediment were sampled. The purpose of this investigation was to develop a quantitative method for detecting enteroviruses in marine sediments so that their relative proportion to viruses freely suspended in estuarine water could be more accurately determined. Poliovirus was found to adsorb readily to natural marine sediments collected along the Texas Gulf coast. A number of substances were evaluated for their ability to elute adsorbed viruses. A solution of 10% fetal calf serum adjusted to pH 10.5 and 0.05M ethylenediaminetetraacetate (pH 11.0) were found to be the best eluents. Using ethylenediaminetetraacetate as an eluent, it was possible to elute virus from large volumes of sediment and reconcentrate the sediment eluate into an economically assayable volume (30 to 50 ml). Poliovirus could be recovered from the sediment with an overall efficiency of 50%. This method was found to be satisfactory for the recovery of naturally occurring animal viruses in estuarine sediments from the upper Texas Gulf coast.     

Development of quantitative methods for the detection of enteroviruses in sewage sludges during activation and following land disposal.

The development and evaluation of methods for the quantitative recovery of enteroviruses from sewage sludge are reported. Activated sewage sludge solids were collected by centrifugation, and elution of the solid-associated virus was accomplished by mechanical agitation in glycine buffer at pH 11.0. Eluted viruses were concentrated either onto an aluminum hydroxide floc or by association with a floc which formed de novo upon adjustment of the glycine eluate to pH 3.5. Viruses which remained in the liquid phase after lowering the pH of glycine eluate were concentrated by adsorption to and elution from membrane filters. The method of choice included high pH glycine elution and subsequent low pH concentration; it yielded an efficiency of recovery from activated sludge of 80% for poliovirus type 1, 68% for echovirus type 7, and 75% for coxsackievirus B3. This method was used to study the survival of naturally occurring virus in sludge at a sewage treatment plant and after subsequent land disposal of the solids after aerobic digestion. Reduction of enterovirus titers per gram (dry weight) of solids were modest during sludge activation but increased to a rate of 2 log 10/week after land disposal.     

Development of quantitative methods for the detection of enteroviruses in sewage sludges during activation and following land disposal.

The development and evaluation of methods for the quantitative recovery of enteroviruses from sewage sludge are reported. Activated sewage sludge solids were collected by centrifugation, and elution of the solid-associated virus was accomplished by mechanical agitation in glycine buffer at pH 11.0. Eluted viruses were concentrated either onto an aluminum hydroxide floc or by association with a floc which formed de novo upon adjustment of the glycine eluate to pH 3.5. Viruses which remained in the liquid phase after lowering the pH of glycine eluate were concentrated by adsorption to and elution from membrane filters. The method of choice included high pH glycine elution and subsequent low pH concentration; it yielded an efficiency of recovery from activated sludge of 80% for poliovirus type 1, 68% for echovirus type 7, and 75% for coxsackievirus B3. This method was used to study the survival of naturally occurring virus in sludge at a sewage treatment plant and after subsequent land disposal of the solids after aerobic digestion. Reduction of enterovirus titers per gram (dry weight) of solids were modest during sludge activation but increased to a rate of 2 log 10/week after land disposal.