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 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%.     

Thermostabilization of enteroviruses by estuarine sediment.

The effect of estuarine sediment on the thermoinactivation of poliovirus type 1 and echovirus type 1 was evaluated. Poliovirus survival was prolonged at 24 and 37 degrees C but not at 4 degrees C in the presence of sediment over the time periods observed. Further inactivation studies were performed at 50 and 55 degrees C to maximize the thermal effects, and similar protection was observed. The supernatant fluid from a mixture of seawater and sediment lacked the protective effect against thermoinactivation, suggesting that prolonged virus survival in the presence of sediment was due to adsorption to particulates. From these observations, it appears that the adsorption of enteroviruses to estuarine sediments may play a significant role in protecting them against thermoinactivation.     

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.     

Thermostabilization of enteroviruses by estuarine sediment

The effect of estuarine sediment on the thermoinactivation of poliovirus type 1 and echovirus type 1 was evaluated. Poliovirus survival was prolonged at 24 and 37 degrees C but not at 4 degrees C in the presence of sediment over the time periods observed. Further inactivation studies were performed at 50 and 55 degrees C to maximize the thermal effects, and similar protection was observed. The supernatant fluid from a mixture of seawater and sediment lacked the protective effect against thermoinactivation, suggesting that prolonged virus survival in the presence of sediment was due to adsorption to particulates. From these observations, it appears that the adsorption of enteroviruses to estuarine sediments may play a significant role in protecting them against thermoinactivation.     

Stability of human enteroviruses in estuarine and marine waters.

Studies of the effects of temperature and salinity on the survival of three enteric viruses (poliomyelitis type 1, echovirus-6, and coxsackievirus B-5) under controlled laboratory conditions and in situ indicate that temperature rather than salinity is the critical factor affecting their stability, in that the higher the temperature the more rapid was the loss of viral infectivity. In the laboratory studies, all three viruses were quite stable at 4 degrees C, with infectious virus still detectable after 46 weeks of incubation. In situ studies on virus survival in free-flowing estuarine or marine waters showed that, although the viruses were more labile in natural waters than in the laboratory studies, they persisted for several months, in some cases during the winter months. At all temperatures and salinities, coxsackievirus B-5 was the most stable, echovirus-6 was intermediate, and poliovirus 1 was the least stable of the viruses tested.     

Stability of human enteroviruses in estuarine and marine waters.

Studies of the effects of temperature and salinity on the survival of three enteric viruses (poliomyelitis type 1, echovirus-6, and coxsackievirus B-5) under controlled laboratory conditions and in situ indicate that temperature rather than salinity is the critical factor affecting their stability, in that the higher the temperature the more rapid was the loss of viral infectivity. In the laboratory studies, all three viruses were quite stable at 4 degrees C, with infectious virus still detectable after 46 weeks of incubation. In situ studies on virus survival in free-flowing estuarine or marine waters showed that, although the viruses were more labile in natural waters than in the laboratory studies, they persisted for several months, in some cases during the winter months. At all temperatures and salinities, coxsackievirus B-5 was the most stable, echovirus-6 was intermediate, and poliovirus 1 was the least stable of the viruses tested.     

Concentration and purification of enteroviruses by membrane chromatography.

A simple procedure for the concentration and partial purification of enteroviruses from tissue culture harvests is described. After removal of acid-precipitating components with a cationic detergent, the detergent and most membrane-coating components were removed by treatment with a cationic-exchange resin. The resin effluent was then acidified, and the virus was adsorbed to epoxy-fiberglass membranes. Virus was then eluted with pH 11.5 glycine-NaOH buffer. Since this eluate contains no orgcentrated simply by acidifying the eluate and passing it through a smaller membrane than that used for the first concentration. As high as 500-fold concentrations can be achieved, with a high efficiency of recovery.     

Concentration and purification of enteroviruses by membrane chromatography.

A simple procedure for the concentration and partial purification of enteroviruses from tissue culture harvests is described. After removal of acid-precipitating components with a cationic detergent, the detergent and most membrane-coating components were removed by treatment with a cationic-exchange resin. The resin effluent was then acidified, and the virus was adsorbed to epoxy-fiberglass membranes. Virus was then eluted with pH 11.5 glycine-NaOH buffer. Since this eluate contains no orgcentrated simply by acidifying the eluate and passing it through a smaller membrane than that used for the first concentration. As high as 500-fold concentrations can be achieved, with a high efficiency of recovery.     

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.     

Modified membrane-filter procedure for concentration of enteroviruses from tap water.

Enteroviruses added to 114 liters of dechlorinated tap water were recovered in a 16-ml sample by a two-stage concentration procedure in which different types of membrane filters were used in each concentration stage. Viruses in tap water at pH 3.5 were first adsorbed to 10-in. (ca. 25.4-cm) epoxy-fiber glass filters (Filterite). Viruses adsorbed to these filters were eluted with a solution of 0.2 M sodium trichloroacetate buffered at pH 9 with 0.2 M lysine. Viruses in this solution were adsorbed to 47-mm asbestos filters (Seitz) without pH adjustment or other modification of the solution. Viruses were recovered from the Seitz filters with 16 ml of either Casitone or fetal calf serum at pH 9. With these procedures ca. 45% of several types of enteroviruses added to 114 liters of tap water could be recovered in the final 16-ml sample.     

Role of sediment in the persistence of enteroviruses in the estuarine environment.

The survival of four enteroviruses commonly found in sewage effluents was examined when the viruses were adsorped to marine sediments in estuarine water and compared with virus survival in estuarine water alone. Echovirus 1, coxsackieviruses B3 and A9, and poliovirus 1 survived longer when associated with marine sediment. When the estuarine water was polluted with secondarily treated sewage effluent, virus survived for prolonged periods in sediments, but not in the overlaying estuarine water.     

Role of sediment in the persistence of enteroviruses in the estuarine environment.

The survival of four enteroviruses commonly found in sewage effluents was examined when the viruses were adsorped to marine sediments in estuarine water and compared with virus survival in estuarine water alone. Echovirus 1, coxsackieviruses B3 and A9, and poliovirus 1 survived longer when associated with marine sediment. When the estuarine water was polluted with secondarily treated sewage effluent, virus survived for prolonged periods in sediments, but not in the overlaying estuarine water.     

Evaluation of MK filters for recovery of enteroviruses from tap water.

The MK filter is an electropositively charged filter that can be used to concentrate enteroviruses from large volumes (400 to 1,000 liters) of water. This filter is less expensive than the commonly used 1MDS electropositive filter. In this study, we compared the recovery of poliovirus 1 (PV1) and that of coxsackievirus B3 (CB3) from 378 liters of tap water, using both the MK and the 1MDS filters. Viruses were eluted from the filters with 3% beef extract buffered with 0.05 M glycine (pH 9.5) and reconcentrated via organic flocculation. At high virus inputs (approximately 10(6) PFU), the overall recovery (after elution and reconcentration) of PV1 and CB3 from tap water with the MK filter was less than that achieved with the 1MDS filter (P < 0.05). The recoveries of PV1 from tap water with the MK and 1MDS filters were 73.2% +/- 26% (n = 5 trials) and 90.2% +/- 5.9% (n = 5 trials), respectively. The recoveries of CB3 from tap water with the MK and 1MDS filters were 32.8% +/- 34.5% (n = 4 trials) and 95.8% +/- 12.0% (n = 4 trials), respectively. This study indicated that the MK filter consistently provided lower recovery, with wider variability, of PV1 and CB3 from tap water than the 1MDS filter.     

Concentration of rotavirus and enteroviruses from blue crabs (Callinectes sapidus)

A simple method for concentration and detection of rotavirus and enteroviruses in the blue crab is described. Virus was separated from tissue homogenates at pH 9.5, concentrated by adsorption to protein precipitates at pH 3.5, and recovered by elution of precipitates at pH 9.2. Test samples of 12 to 15 ml were produced from an initial 100 g of crab tissues. Cat-floc precipitation was used to remove sample toxicity for cell cultures. Recovery effectiveness averaged 52% with poliovirus 1, echovirus 7, and coxsackievirus B5 and 23% with rotavirus SA11.     

Concentration of rotavirus and enteroviruses from blue crabs (Callinectes sapidus).

A simple method for concentration and detection of rotavirus and enteroviruses in the blue crab is described. Virus was separated from tissue homogenates at pH 9.5, concentrated by adsorption to protein precipitates at pH 3.5, and recovered by elution of precipitates at pH 9.2. Test samples of 12 to 15 ml were produced from an initial 100 g of crab tissues. Cat-floc precipitation was used to remove sample toxicity for cell cultures. Recovery effectiveness averaged 52% with poliovirus 1, echovirus 7, and coxsackievirus B5 and 23% with rotavirus SA11.