DETECTION OF VIRUSES IN WATER A REVIEW OF METHODS AND APPLICATION William F. Hill, Jr., Elmer W. Akin, and William H. Benton ENVIRONMENTAL PROTECTION AGENCY Water Quality Office Division of Water Hygiene Gulf Coast Water Hygiene Laboratory Dauphin Island, Alabama 36528 1971 ------- Copyright(Cy1971. Reprinted by permission of the Board of Trustees of the University of Illinois from PROCEEDINGS THIRTEENTH WATER QUALITY CONFERENCE February 1971 VIRUS AND WATER QUALITY: OCCURRENCE AND CONTROL Edited by Vernon L. Snoeijink Department of Civil Engineering University of Illinois Environmental Protection Agency of the State of Illinois ------- DETECTION OF VIRUSES IN WATER: A REVIEW OF METHODS AND APPLICATION WILLIAM F. HILL, JR., ELMER W. AKIN, AND WILLIAM H. BENTON Environmental Protection Agency, Water Quality Office, Division of Water Hygiene, Gulf Coast Water Hygiene Laboratory, Dauphin Island, Alabama 36528. ABSTRACT One of the major problems facing environmental health officials in regard to water quality is related principally to the unavail- ability of reliable and standard methods to concentrate, detect, and isolate low-multiplicities of virus from very large volumes of water. The critical examination of all water supplies for the presence of viruses (including waters used for drinking, recreation, and food production) requires a quantitative approach. In order to be quanti- tative, measurable quantities of water must be examined. This is the only way in which a definitive assessment can be made as to the dis- tribution and extent of virus contamination of our water resources. The challenge to the virologist is related to the need for developing new and/or improved techniques in the laboratory that have a high likelihood for adaptation to the real world situation. In this regard, a number of techniques have been shown experimentally to be good can- didates for assessing the occurrence of viruses in various types of water. The most promising methods are: (i) membrane-adsorption technique; (ii) adsorption to precipitable salts, iron oxide, and polyelectrolytes; (iii) aqueous polymer two-phase separation technique; and (iv) soluble alginate filter technique. Most of these methods have shown good-to-excellent virus recovery efficiencies as well as a reasonable efficacy for concentrating viruses from water in controlled laboratory experiments. Other methods such as (i) continuous-flow ultracentrifugation; (ii) forced-flow electrophoresis and electro- osmosis, and (iii) hydroextraction have also shown favorable virus recovery efficiencies under laboratory-controlled conditions but fall short as candidate techniques for real world virus-in-water problems. From the data, it would appear that the most promising methods for detecting and isolating low-multiplicities of virus in clean and finished waters are those that rely on virus adsorption and/or retention coupled with a flow-through sampling system. For waters that are mod- erately or grossly turbid, it would appear that aqueous polymer two- phase separation may be the better approach. In this review paper, the above methods are briefly described in terms of mechanisms, procedure, and efficiency. The methods are evaluated in terms of speed, simplicity, and economy of application. ------- INTRODUCTION Environmental health security as related Co our water re- sources is seriously jeopardized by the lack of standardized methodologies for detecting viruses in water, The unequivocal need for adequate and standardized virus methods for determining the occurrence of viruses in water and waterways has been prof- fered for a number of years (Public Health Service, Drinking Water Standards, 1962; Kramer, 1965; Berg, 1967), According to the Com- mittee on Environmental Quality Management of the Sanitary Engineer- \ itig Division of the American Society of Civil Engineers, "the in- adequacy of methods for detecting, identifying, and enumerating viruses in samples of water constitutes an important Iserious] gap in water quality control" (Committee report, 1970), The lack of a method for the detection, of viruses in water then would seem to be an enigma. Oversimplified, the total problem can be reduced to the unavailability of reliable and standard methodology to sample, recover, concentrate, and isolate viruses occurring at low-multi- plicity levels in waters. There are a number of aspects of the problem none of which are mutually exclusive. One aspect is con- cerned simply with methods for sampling and detecting viruses in water, Tor example, it is well documented that detecting viruses in grossly polluted waters (sewage effluents) by the gauze-pad technique has been phenomenally successful not because the technique is particularly efficient but rather because of the high-multiplicities ------- -2 of viruses that occur in fecally polluted waters. Another aspect of the problem is two-fold and is concerned with methods for quantitative assessment of viruses in water. This involves (i) collecting samples of water of sufficient volume which would have a high likelihood of containing viruses, and (ii) concentrating the viruses occurring at low-multiplicities therein for isolation. It is the selection of a suitable method for concentrating viruses from very large volumes of water that represents the greatest challenge to a virological methods research activity. Consequently, the greatest need in virus methods research is techniques that efficiently concentrate viruses from very large volumes of water regardless of whether the water is used for drinking, recreation, or food production. Unfortunately, a reliable method for concen- trating low-multiplicities of virus from large quantities of water of the real world is not readily available for routine use, A third aspect of the problem for detecting viruses in water is con- cerned with laboratory isolation, identification, and enumeration of recovered viruses. This requires the selection of susceptible virus-host systems; e.g., a number of different cell cultures and/ or laboratory animals that will support the propagation of the various virus isolants. Since 100 or more virus serotypes are shed by the fecal route and therefore can be expected to occur in domestic wastes and waterways, this is not a simplistic task. The ------- -3 availability of facilities and trained personnel to handle primary- cell cultures, continuous-cell cultures, and suckling mice would be mandatory. The final determinations of the laboratory require- ments, however, would have to be made by a professional virologist. In summary, one of the major problems facing environmental health officials in regard to water quality control is related principally to the unavailability of reliable and standard methods to concentrate, detect and isolate low-multiplicities of virus from very large volumes of water. The critical examination of all water supplies for the presence of viruses (including waters used for drinking, recreation, and food production) requires a quantitative approach. And, in turn, the challenge to the virologist is related principally to the need for developing new and/or improved tech- niques in the laboratory that have a high likelihood for adaptation to the real world situation. In order to be quantitative, measur- able quantities of water must be examined. The smallest quantity of water considered adequate for detecting low-multiplicities of virus is probably 100 gallons (378.5 liters) for clean or finished waters. For waters that are moderately or grossly polluted, a minimum sample size of at least 1 liter or perhaps 1 gallon (3,78 liters)may be adequate. The point of departure of this paper con- cerns methods that show promise for concentrating viruses from very large volumes of water. The specific purpose of this paper is ------- -r-3a to describe and review a particular method and then expound briefly on the advantages and disadvantages of that method in terms of efficiency and application. ------- -4 BACKGROUND APPROACH TO THE PROBLEM The approach to the methods research problem for concentra- ting viruses from water stems from the physicochemical properties of the virus particle itself and is related principally to those procedures applicable to macromolecular proteins. "For example, viruses are nucleoproteins and behave as colloidal hydrophilic particles in suspension. Consequently, many of the physical and chemical properties of viruses identify with the properties common to proteins. In this regard, viruses manifest properties of solu- bility which decrease with increasing concentrations of very soluble salts such as ammonium sulphate. Viruses are amphoteric; i.e., capable of reacting either as electro-positive or as electro-negative. They also have determinable isoelectric points (electro-neutral), Since viruses exhibit polarity they are also immiscible in organic solvents such as diethyl ether, n-butanol, chloroform, and fluoro- carbons. Virus particles manifest unique surface properties as exhibited in their ability to adsorb readily to a number of sub- stances such as celite, alumina gel, tricalcium phosphate, starch, and various resin and cellulose derivatives (Schwerdt, 1965), Viruses have measurable molecular weights and sizes. For example, a poliovirus virion has a molecular weight of 7 x 10® daltons and is approximately 28 nm in diameter. Consequently, viruses also lend themselves to sedimentation from solutions by ultracentrifugation techniques. ------- -5 With regard to the nature of viruses, a number of reported techniques have been shown experimentally to be good candidates for assessing the occurrence of viruses in various types of water. Likewise, from field studies, a number of these techniques have also shown promise for adaptation to the real world situation. The most promising techniques for concentrating viruses from water involve, primarily, modifications of methods the principles of which have been known for some time. For example, modification of methods such as; (i) membrane-adsorption technique, (ii) adsorption to precipitable salts, iron oxide and polyelectrolytes, (iii) aqueous polymer two-phase separation technique, (iv) soluble alginate filter technique, (v) continuous-flow ultracentrifugation, (vi) forced-flow electrophoresis and electro-osmosis, and (vii) hydroextraction procedures, are all considered potentially adaptable to concentrating viruses from water. From a viruses-in-water standpoint, many of the above methods are still developmental in that they have been studied under rather rigidly controlled labo- ratory conditions. Nevertheless, some of these methods are under- going field evaluation by being applied, on a limited scale, to suspect virus-contaminated raw water supplies. Ideally, an acceptable method should satisfy the basic criteria of a good method by being simple, rapid, sensitive, and reliable. In addition, an acceptable method must be economical from an application standpoint. Some methods that have shown good ------- -6 promise in controlled laboratory experiments, unfortunately, fall short when they are applied to the field,, For example, most of the methods mentioned above show good to excellent virus recovery efficiencies as well as a reasonable efficacy for concentration of virus from water in laboratory-controlled experiments. However, the water sample size which has ranged from 10 ml up to 19 liters has been most frequently used for the evaluation studies regardless of whether the work was conducted in the field or in the laboratory. Consequently, the adaptation of s'ome of these methods to very large quantities of water of at least 100 gallons remains mere conjecture. This is important, for if viruses occur at low-multiplicities in clean and/or finished vraters then the expectation of success for their detection and isolation is limited primarily by the sample size, and for finished waters 100 gallons is considered minimal. For moderately polluted waters, sample sizes of 1 liter to 1 gallon may be sufficient as mentioned previously. ------- -7 QUANTIFICATION OF VIRUSES AND ENUMERATIVE RELIABILITY There are two methods used to quantify or titer viruses; the quantal assay technique and the plaque assay technique. The one method which yields a quantal response (all-or-none) is commonly called, in virology, the TCID^q assay method. The"ICID^q" designation refers to tissue culture infective dose 50 percent. The expression for this method can vary depending upon the host and the indicated response; i.e., lethal dose 50 percent (LD^q); egg infective dose 50 percent (EID^q); and effective dose 50 percent (ED50), to name a few. Briefly, the tissue culture procedure con- sists of making serial logarithmic dilutions of the material to be assayed and then inoculating a number of tubes (usually 4 or 5 tubes per dilution) containing cell-monolayers. Following an in- cubation period, the occurrence or absence of cytopathlc effect (CPE) in the cells of each tube is observed microscopically. The 50 percent endpoint may then be calculated by using the method of Reed and Muench (1938) or some other acceptable method. The ex- pected reproducibility of the quantal assay method is within + 0.3 log. The lack of precision of this assay method should be recognized and caution exercised not to credit 50 percent endpoint determinations with a precision they do not have; a 1.5- or 2-log difference in titer is usually considered significant while a 1-log (or less) difference may be equivocal. The quantal assay ------- -8 method also permits the application of the statistical most- probable-number (MPN) method for estimating virus multiplicities (Chang, et at, 1958). The precision and accuracy of the adapted MPN method is predicated primarily upon the dilution interval and the number of tubes inoculated per dilution. The other method is called the plaque assay method. This method is considered by virologists to be the most precise method to quantify viruses since a plaque or plaque-forming unit (PFU) can be initiated by a single virion. Plaques appear as microscopic circumscribed areas of cytopathology which result from the contig- uous spread of virus in susceptible cells under a semi-solid overlay (Figure 1). Some viruses do not produce plaques, however, and some viruses exhibit a low-plaquing efficiency. However, when a virus does possess a high-plaquing efficiency, it is inexcusable to use any other method for quantification. Additionally, from the standpoint of enumerative reliability, it is also known that single virions as well as virion aggregates both possess the capac- ity to produce single plaques. Consequently, it should be recognized that factors, such as: (i) the number of virion aggregates; (ii) the size of aggregates; and (ill) the aggregate-single virion ratio in a given suspending medium contribute to the total enumerative error term. Any virus handling techniques that enhance aggregation or promote disaggregation should be recognized in order to maintain enumerative reliability. In our laboratory, we found that selection ------- FIGURE 1. Viral plaques produced in cell-monolayers of HEp-2 cells; from left to right: echovirus type 6; coxsackievirus B—1; and poliovirus type 1 (LSc2ab). ------- -9 of a suitable plaque assay diluent can be important under certain circumstances (Hamblet, et al. 1967). A typical experiment is shown in Table 1 where it may be observed that the plaquing effi- ciency of poliovirus type 1 was increased almost 5-fold simply by diluting the virus in nutrient broth when compared to diluting the virus in Hanks' balanced salt solution. Furthermore, there is a statistical notation that must be considered when quantitative virus determinations are made by the plaque technique; i.e., the number of plaques produced on a cell-monolayer follows a Poisson distribution; assuming no error other than random sampling error (Larson and Reinicke, 1965), Since enumerative reliability is closely allied with the Poissonian statistical inference, experi- mental designs must include a replicate sampling technique. The analytical examination of these multiple observations (replicate PFU counts) by statistical methods can then serve as a basis for making valid interpretations of quantitative experiments. Data handled in this manner will control and should ultimately minimize enutneratlve error by measuring variability. It should be obvious, however, that replicate sampling can only be expected to narrow the margin of error not eliminate error. Under conditions of good analytical control, the 95 percent fiducial confidence limits for a single virus infectivity assay, as determined by replicate sam- pling, are frequently greater than + 10% and may approach + 50% of the mean plaque count. Moreover, the? expected error, based on the ------- TABLE 1 EFFECT OF PLAQUE ASSAY DILUENT ON POLIOVIRUS MULTIPLICITY ASSESSMENT FROM EQUAL VIRUS POOLS Replicate samples HBSS - Poliovirus poola NB - Poliovirus noolk PFV/ml PFU/ml 1 470 580 270 2200 1100 2200 2 590 460 640 2500 2400 1600 3 440 510 460 2100 1600 3500 4 420 510 450 2400 3100 3100 5 540 430 480 2700 1400 2700 Mean + SE 483 + 22 2307 + 174 95% CI 435 to 532 1932 to 2681 Coefficient of variation 18% 29% £ HBSS, Hanks' balanced salt solution. ^NB, Nutrient broth (Difco). ------- -10 coefficient of variation, of replicate plaque counts usually ranges from 10 to 30% and should not normally exceed 50 percent. The precision and accuracy of the plaque assay method is generally accepted to be of a high order. Nevertheless, if the concept of enumerative reliability is to be maintained, then ran- dom error must be included as one of the parameters inherent in counting virus plaques. Furthermore, an awareness of the expected variability incident to estimating virus multiplicities by replicate plaque assays is mandatory for the professional virologist. The interpretation of quantitative virological data must be meticulously sophisticated and utmost caution exercised by the novice not to consider single (or duplicate) plaque counts as absolute numbers. This is particularly important when methods research concerns quantitative virus assessments. In this regard, placing too much emphasis on the percent of virus recovered, although mathematically accurate, can often lead to not only distorted but meaningless value judgments. For example, the interpretation of virus recovery data when the initial input-multiplicity of virus is high (2 x 10^ to 2 x 10® PFU) or low (2 to 4 PFU), on the basis of percent recovery, may be completely misleading because of the random error term and the expected variability of enumerating viruses by the plaque tech- nique, Consequently, to evaluate the efficacy of a virus concen- tration method in a typical virus-input virus-output experiment, the concentration of virus infectivity (in PFU) observed coupled with ------- -11 percent recovery are both important. The percent of virus recovered as a single parameter may be nothing more than ancillary information and in repeat experiments may often range, conservatively, from 20 to 90 percent and occasionally even exceed 100 percent; a calculated artifact. In summary, the plaque assay technique is the most precise method for enumerating virus multiplicities, and as such is the preferred quantitative method for laboratory-controlled experiments. The quantal assay technique although lacking enumerative precision * is considered by many virologists to be more sensitive for detecting very low (<1 PFU/ml) multiplicities of virus and, obviously, the only technique applicable to virus detection for viruses that do not form plaques. The quantal assay procedure would also be the preferred detection method for mixed virus populations present in a single sample. For best results in field trials, both the plaque assay and the quantal assay techniques should be conducted in parallel on all samples. ------- -12 SAMPLING WATER FOR VIRUS ASSESSMENT Fundamentally, there are only two methods available for sam- pling water for the presence of viruses. One method is an in eitu entrapment technique and is called the gauze-pad or swab method. This method is strictly qualitative and consists of suspending a gauze pad (filled with cotton or other material) in the water to be examined for a period of time, usually 24 hours to several days. The pad is then treated with 1 N sodium hydroxide (pH8+) to enhance elution of any 4entrapped viruses and the fluid expressed from the pad. The other method is simply a water collection technique and is called the grab- or dip-sample method. This method is quanti- tative and consists of dipping a bottle or jug in the water to be examined. Both methods have been used in the field, with varying degrees of success, for detecting the presence of viruses in raw waters. Some of the obvious advantages and disadvantages of both methods have been reviewed recently (Grabow, 1968). It should be pointed out that the efficacy of both methods is closely linked with the nature of the water source. For example, when either or both methods were applied by a number of workers to waste-waters, sewage effluents, and other bodies of water that were grossly pol- luted with treated or untreated domestic wastes, a nun^er of suc- cessful virus isolations were made (Chin, et al, 1967; England, et al. 1967). However, when the two methods have been tested or ------- -13 compared in parallel, the gauze-pad technique has been shown to be the superior virus detection method. It should be noted that dip-samples usually consist of 100 to 200 ml of sample and there- fore bias does enter into the evaluation* Some typical observa- tions are shown in Table 2. In summary, if the water to be examined for viruses is grossly polluted with fecal wastes then the application of the gauze-pad technique will provide a high likelihood for virus detection. It must be emphasized that the findings, however, will be strictly qualitative not quantitative. If quantitative virus assessment of waters is desired then the dip-sample method, or an equivalent modification of some technique, where the volume of water is measured, must be used. It should be noted here that it is only the quantitative approach that will definitively assess the dis- tribution and extent of virus contamination in our waterways (including surface waters as veil as reclamation water and finished drinking water) and thereby permit meaningful conclusions to be made. Value judgements based on qualitative data will not resolve the public health management of virus-in-water problems nor elucidate the potential health threat of water-borne viruses and the epidemi- ology of viral diseases transmitted by the water route. ------- TABLE 2 COMPARISON OF GAUZE-PAD AND DIP-SAMPLE METHODS FOR ISOLATING VIRUSES FROM SEWAGE Virus Recovered Reference Gauze- Number positive "Number tested -Pad % Positive Dip-Sample Number positive Number tested % Positive Melnick et at, 1954 196/324 60.4 66/231 28.6 Mack et at, 1958 , 66/602 10.9 26/651 4.0 Bloom et at, 1959 61/462 13.2 17/368 4.6 Mack et at. 1962 44/268 16.4 1/293 0.3 Lund and Hedstrom, 1969 74/84 88.1 32/84 38.1 ------- -14 METHODS FOR CONCENTRATING VIRUSES IN WATER Within the past several years, a number of reports have appeared in the literature on methods for concentrating viruses from water. Many of these methods show sufficient promise to permit the quantitative assessment of viruses in waters of the real world. Some of these methods have been mentioned previously in this paper and have also been reviewed recently (Berg, 1967; Grabow, 1968). These same methods were considered by the Committee on Viruses in Water (Committee Report, 1969) to merit continued investigation. In this section, a particular method will be briefly described and then evaluated as to the advantages and disadvantages of that method in terms of efficiency and application. Merribrarie-adsprption technique. The mechanism of the membrane-adsorption technique is related principally to the unique surface properties of the virion; i.e., under specified conditions, viruses efficiently adsorb to a variety of substances including microporous membranes. These membranes which are composed of cellulose derivatives are commonly used to partially purify or clarify crude virus-cell harvest material by filtration. Membranes have also been used for sizing viruses (Ver, et al, 1968). In virus filtration experiments, any observed losses in virus titer in the filtrates after filtration have been attributed ------- to (i) the presence of virion aggregates, and (ii) adsorption of virions to the matrix of the menbrane. The concentration of viruses by the membrane-adsorption tech- nique depends on (i) adsorbing viruses to the membrane, and (ii) then removing the viruses from the membrane by elution. Elution is usually carried out at a high pH; i.e., pH 8 to 9. Substances used to elute viruses from the -embrane are usually proteinaceous in nature; i.e., whole serum, albumin, beef extract, casein, veal infusion broth, and nutrient broth. Some of these substances have also been used to pretreat membranes which enhances virus filtra- tion efficiency. Many of the factors that influence the filtration of viruses through the matrices of microporous membranes have been described by Cliver (1965). Interestingly, Cliver also observed that viruses adsorb to the membrane unless the membranes were pretreated with serum or a gelatin solution, Cliver (1965) indi- cated that whether virus passed through or failed to pass through the filter membrane was probably the net result of several processes i.e., (i) adsorption of virus to the matrix surfaces of the membrane (ii) competition of macromolecules for adsorption sites on the mem- brane, and (iii) the presence or absence of viral aggregates, to name a few. In studies concerned with virus interactions with mem- brane filters, Cliver (1968) su—larized that loss of virus in membrane filtration was primarily due to adsorption of the virus to ------- -16 the membrane matrix surfaces. In turn, virus adsorption phenomena was shown to be influenced by; (i) chemical composition of the filter membrane, (ii) ratio of pore diameter to the diameter of the virion, and (iii) the absence of substances such as those occurring in serum that interfere with virus adsorption. In this regard, cellulose triacetate membrane filters were shown to adsorb virus poorly when the porosity exceeded the virion diameter by as much as 3 times. Conversely, cellulose nitrate membrane filters were shown to adsorb virus very efficiently even when the porosity exceeded the virion diameter by as much as 285 times (Cliver, 1968). Working with virus- seeded 1-liter samples of Chicago tap-water, Cliver (personal Qonmv.rsl- aticmi 1970) indicated that he achieved about 260-fold virus concen- tration and virus recoveries of 26 to 90% with the membrane-adsorption technique. Concentration of enteroviruses on membrane filters from crude virus-cell harvest material was investigated by Wallis and Melnick (1967a), They also observed that enteroviruses can be made to adsorb or to pass through membrane filters simply by manipulating the virus suspending medium. For example, the addition of salts to the virus suspending medium, particularly salts containing divalent cations such as MgC^ were shown to significantly enhance viral adsorption. Additionally, the adjustment of the virus suspending medium to pH 5 (close to the isoelectric point of poliovirus type 1) was shown to ------- -17 markedly enhance viral adsorption. Conversely, the presence of organic and proteinaceous substances in the virus suspending medium was observed to interfere significantly with virus adsorp- tion to the membrane matrices, presumably by competing for membrane adsorption sites. They referred to these viral adsorption inter- fering substances as membrane-coating components or simply as MCC. These same authors reported that MCC was effectively removed by passing the liquid sample through an anion-resin column [Dowex 1-X8 (Cl~) 100-200 mesh]. Other workers (Borneff, 1970; Schafer, 1970), however, have had less than success in removing MCC in natural waters with anion-resins and as a result have apparently abandoned the use of microporous membranes for virus concentration and detection in raw waters in Germany. Nevertheless, Wallis and Melnick (1967a) state that with the use of membrane filters, virus can be quanti- tatively recovered from crude virus-cell harvests and 80- to 100-fold concentration of virus achieved. These investigators used serum as the eluent. The membrane-adsorption method has also been applied to field samples for concentration and detection of viruses from sewage (Wallis and Melnick, 1967b). The method was tested in Houston, Texas over a period of seven months. Briefly, samples which consisted of 1-gallon (3.78 liters) quantities of raw sewage were initially clar- ified by prefiltration and then pressure-filtered through a 47-mm or 90-mm HA Millipore membrane (0.45 ym porosity). During the period ------- -18 of the study, a total of 2,795 isolants were recovered from 10 separate samples (Range: 10 PFU to 701 PFU/gallon). These results were compared with those observed from small samples of unconcentrated sewage collected at the same time when only 4 isolants were recovered. Rao and Labzoffsky (1969) investigated the efficacy of mem- brane filters to detect low-multiplicities of viruses in what they considered to be large volumes of surface waters (500-ml quantities). They combined an AP 25 MF prefilter and an HA Millipore membrane filter (0.45 vm porosity) as a single unit. Any virus adsorbed on the prefilter was also eluted with a 3% beef extract, pH 8 eluent ¦in situ in a single operation. These same investigators indicated that virus trapped in the prefilter was lost if prefiltration was used to clarify the water as a preliminary step. These investiga- tors observed experimental virus recovery efficiencies of 53 to greater than 100 percent. They also determined the necessity of having electrolytes (salts) such as CaC^ in the water to enhance viral adsorption to the membranes. In their experimental studies, they added 200 ppm Ca^+ to the raw water before conducting the experiments. Moore, et at. (1970) compared'various applications of a modi- fied membrane-adsorption technique for concentration of viruses in waste-water. They also observed that the apparent occurrence of ------- -19 KCC In waste-water adversely affected the efficiency of the mem- brane filters (0.45 ym porosity) to adsorb poliovirus. They suc- cessfully removed MCC from waste-water with the anion-resin pre- treatment as described by Wallis and Melrtick (1967a) but failed to remove MCC with a preliminary protamine sulfate flocculation tech- nique. Experimentally, Moore and associates (1970) found that the combination of aluminum hydroxide flocculation followed by membrane filtration (retention of the virus-aluminum hydroxide complex) gave the best results showing 81 to 100^ recovery of input (seeded) poliovirus. These same investigators showed that the combination of protamine sulfate flocculation followed by filtration through an AP 20 MF prefilter and then concentrating the virus on a Millipore membrane filter yielded a virus recovery of only 26 to 31 percent. Hill, et al% (unpublished data) are currently evaluating the adaptability of the inillitube MF cartridge filter (0.45 ym porosity) to concentrate virus from very large volumes of water of 100 gallons. Their preliminary findings indicated thct poliovirus can be concen- trated up to 140- to 218-fold from demineralized-water and raw estuarine-water (salinity 25 °/oo)( respectively. Virus recovery from the demineralized-water was 56% while virus recovery from the raw estuarine-water was 79 percent. Some virus penetrated the filter when raw estuarine-water was used. In these studies, the pH of the 100-gallon water samples was adjusted to pH 4,5 before filtration. ------- -20 Divalent cations were added to the demineralized-water (500 ppm Ca^+). No salts were added to the estuarine-water. Elution of virus from the cartridge membranes was achieved by using 5 X nutrient broth (Difco), pH 8.5+ as the eluent. Berg (1970) has also been checking out the membrane-adsorption technique for concentrating viruses from large volumes of water. By the use of 1-liter volumes of Mcllvane's buffer (0,05 M Na2HP0^, pH 7,0) seeded with virus (31 to 169 PFU/liter), Berg observed the following virus" recoveries: (i) xeovirus type 1, 52 to 78%, (ii) coxsackievirus B-3, 97%, (iii) echovirus type 7, 121%, and (iv) poliovirus type 1, 103%, In these studies, virus was eluted from the membranes with 3% beef extract and subsequently sonicated before virus assay. In larger volumes of water; i.e., 25 gallons (94,5 liters), virus recoveries have ranged from 50 to 75 percent. Most of the large volume experimental work was done with distilled- water. Virus was not consistently recovered quantitatively when tap water was used, presumedly because of the presence of organic substances competing for virus adsorption sites on the membrane matrix. Joseph (personal aommuniaation3 1970) has used the membrane- adsorption technique in an attempt to isolate viruses from treated drinking waters in Maryland, No virus, however, was isolated from the drinking water source during the period of the study. ------- -21 To summarize, the membrane-adsorption technique for concen- trating and detecting viruses in various types of water holds considerable promise as being an acceptable method. This state- ment is in agreement with the conclusions of the Committee on Environmental Quality Management (1970). Experimentally, volumes of water from 10 nil to 100 gallons (378.5 liters) have been eval- uated by the membrane-adsorption technique for virus concentration and recovery efficiency with favorable results. Virus recovery has ranged from 26% to greater than 100 percent. Equally significant, virus concentration has ranged from 80- to 260-fold. Some typical results are shorn in Table 3, Real world application of the membrane-adsorption technique has been rather limited. Wallis and Melnick (1967b) examined raw sewage as it entered a treatment plant in Houston, Texas. This field test was conducted over a period of seven months. One-gallon size dip-samples were collected once or twice a month and a total of 2,795 isolants were recovered from a total of 10 separate samples. Fundamentally, the membrane-adsorption technique may be con- sidered a simple technique. The efficacy of the technique would seem to be predicated on (i) the proper selection of membrane materials, (ii) the addition or presence of divalent cations, (iii) the adjustment of the suspect virus-contaminated water to pH 5 or below, (iv) the removal of MCC from raw water, and (v) the selection ------- TABLE 3 RECOVERY OF VIRUS FROM WATER BY THE MEMBRANE-ADSORPTION TECHNIQUE Reference Type of water Volume of water Percent of virus recovered Wallis and Melnick, 1967 Sewage 1.5 gallons - 100 Rao and Labzoffsky, 1969 Surface-water 500 ml 53 - 100 Cliver, 1970 Tap-water 1 liter 26 - 90 Moore, et alOJ 1970 Waste-water 1 gallon 81 - 100 Berg, 1970 Distilled-vater 25 gallons 50 - 75 Hill, et al.3 ( ) Estuarine-water 100 gallons 56 - 79 ------- -22 of an efficient eluent. The major advantages of the membrane- adsorption technique are simplicity, speed, and sensitivity. The major disadvantages of the technique are related principally to the types of water to be examined for virus. For example, the occurrence of membrane-coating components in various types of raw waters would adversely affect the efficiency of virus adsorption. In addition, waters that are highly turbid would be expected to clog the filter prematurely. Finally, there are cost consider- ations; routine^ sampling of waters might be considered low to moderate. Initial costs, however, for stainless-steel filter holders capable of processing 100-gallon quantities of water could exceed $1000.00 if more than one holder was required for the water sampling system. Adsorption to preoipitable salts, iron oxide, and poly electrolytes. The mechanism of these techniques is similar to the membrane- adsorption technique in that concentration of virus by the use of precipitable salts, iron oxide, or polyelectrolytes relies on the ability of viruses to adsorb efficiently onto the selected adsorbent. The concentration and purification of viruses by adsor/tion to a variety of adsorbents has been commonly practiced In virology lab- oratories for almost 40 years (Schwerdt, 1965). For example, Sabin ------- -23 (1932) concentrated and partially purified poliovirus by adsorb- ing the virus to alumina gel C and subsequently eluting the virus at high pH. More recently, however, the application of virus adsorption to concentrating viruses from water under experi- mental conditions has been reported to be successful. Stevenson, at. (1956) used an alum flocculation procedure for concentrating coxsackievirus A-2. They added aluminum sulfate under specified conditions to the virus-contaminated water and allowed a floe to form -in situ. Virus was then eluted from the aluminum hydroxide floe at an elevated pH (approximately pH 8+). These investigators indicated that the alum-floc method permitted virus concentrations of 100-fold or greater and was capable of detecting as little as 0.00625 LD^q/0.02 ml (volume inoculated per mouse). Wallis and Melnick (1967c) concentrated a number of different viruses by adsorption on aluminum phosphate, aluminum hydroxide, and calcium hydrogen phosphate floes. In all cases, preformed floes (precipitates) were added to the virus-contaminated aqueous suspensions or sewage effluents. By using conditions found optimal for virus adsorption, they observed that only acid-sensitive viruses were concentrated on the aluminum phosphate floe; e.g., herpesvirus, % influenza, rubella, and vaccinia, to name a few. Conversely, all viruses tested except reovirus and adenovirus were concentrated on the aluminum hydroxide floe and the calcium hydrogen phosphate floe. ------- -24 Among the viruses tested, enteroviruses were also included; e.g., polioviruses, echoviruses, and coxsackieviruses. Noteworthy, adenovirus was adsorbed only to the aluminum hydroxide floe and reovirus was not adsorbed to any of the salts tested. With four of the viruses tested (herpesvirus, measles, poliovirus, and echo- virus), quantitative recovery of the viruses was achieved from 1-liter volumes containing as few as 100 PFU/liter, Recovery of virus exceeded 80% in most cases.. The use of aluminum hydroxide floe as the adsorbent for concentration and detection of viruses occurring naturally in sewage was also undertaken by Wallis and Melnick (1967c). In sewage samples of 1-gallon quantities, 204 isolants were isolated over a period of four weeks. No viruses were isolated from the same samples prior to concentration with the aluminum hydroxide floe procedure. The method is apparently limited, however, by sample size; i.e., being confined to about a 2-gallon quantity, England (personal aomtunication, 1970b) has evaluated, over a three year period, the aluminum hydroxide floe and the calcium hydrogen phosphate floe procedures (modified after Wallis and Melnick, 1967c) for concentrating and Isolating viruses from raw sewage and sewage effluents. Concentration factors up to 100-fold were achieved with the aluminum hydroxide floe procedure. Concen- tration factors up to 300-fold were achieved with the calcium ------- -25 hydrogen phosphate floe procedure. Virus recoveries with both flocculation procedures have ranged from 80 to 100% with adeno- virus and various enteroviruses. The procedures were observed to be less efficient for recovering reovirus. The concentration of virus (coxsackievirus A-9) by adsorption to iron oxide was studied by Rao, et at• (1968). They evaluated a number of different iron oxides and concluded that magnetic iron oxide designated as M, 0. 2530 (available from Magnetic Tape Division of Charles Pfizer Co,) was the best virus adsorbent. They conducted their experiments by passing 500-ml quantities of virus- contaminated water through iron oxide packed in columns containing 25 grams of iron oxide. They eluted the adsorbed virus from the iron oxide in situ with 3% beef extract at pH 8 with an 87 to 90% recovery. The researchers indicated that one of the limitations of the method related to clogging of the iron-oxide bed with sus- pended material. They concluded that the technique exhibited excellent capacity for adsorbing virus and therefore merited addi- tional investigation, particularly in regard to the method's effectiveness for detecting low-multiplicities of virus in large volumes of water, Rao (personal oorrmmioation3 1970) in additional studies, has applied the method experimentally (virus-input studies) to larger quantities of natural waters; i.e., 50 liters of river- water and 150 liters of tap-water. The procedure has also ------- -26 been modified. For example, the natural waters were first clari- fied by filtration through a 47-mm, AP 25 MF fiberglass prefilter pad and then filtered through an iron-oxide bed "sandwiched" between two 47-mm AP 25 MF prefilter pads. Virus adsorbed to the iron oxide was eluted by passing in situ 100 ml of 3% beef extract, pH 8 through the system. Concentration of the virus in the beef extract eluate was also accomplished. This was done by adding 1200 ppm Mg^ (as Mg in MgC^^I^O) to the 100 ml of eluate, adjusting the acidity to pH 3 and then passing the fluid through a 47-mm, 0.45 um porosity Hillipore membrane filter. The adsorbed virus was then again eluted in situ with 5 ml of 3% beef extract at pH 8. By using the two-step adsorption-elution procedure, Rao claims that virus recovery of 100% can be obtained from both types of water. If these results can be confirmed, this two-step procedure may represent a significant breakthrough since the second adsorption- elution step has not been reported previously. Two aspects of these findings are significant; (i) the physical concentration of the system was 30,000-fold for the tap-water and 10,000-fold for the river-water and (ii) the addition of Mg2+ and the adjustment to pH 3 may have solved the problem of interference of virus ^adsorption to membranes as observed by other workers (Moore, at, 1970; Wallis and Melnick, 1967a; Berg, 1970, and Hill, et aX%9 unpublished data). The iron oxide virus-adsorption procedure was not applied ------- -27 to the real world situation by Rao and associates. Jakubowski and Hoff (personal oorrmuni cation, 1970) are currently evaluating iron oxide as a virus adsorbent for adaptation to concen- trating and detecting viruses from tap-water and estuarine-water. They are using a thin-layer filtration procedure in which the iron oxide is suspended in distilled-water and then filtered onto an AP MF fiberglass prefilter pad to form the adsorbing layer. With filter loads of 3 and 4 grams of iron oxide (using the 142-mm Hillipore filtration unit), these researchers have observed virus recoveries of 40 to 64% from 10-liter volumes of tap-water and 22 to 37% from 10-liter volumes of estuarine-water. Metcalf (-personal comrumaabion1970) is also evaluating the efficacy of iron oxide to concentrate and detect viruses from water. By use of the 142-mm Millipore filtration system and the "sandv/ich" technique, this investigator has observed virus recoveries ranging from 80 to 97% in controlled laboratory studies. Additionally, he has indicated that 5 to 10 PFU of enteroviruses can be recovered from volumes ranging from 1 to 10 liters of water by the method. The maximum flux of the iron oxide filtration system was about 5 gallons (18.9 liters) per hour. It was also observed that clogging of the iron oxide filtering surface occurs in the presence of very turbid water (e.g., clay, soil, or silt) and thereby impeded the filtration process and significantly lowered the volume of water ------- -28 capable of filtration. SchSfer and Bomeff (personal communication, 1970) have used a ferric chloride-flocculation method combined with the aqueous poly- mer two-phase separation system for concentrating virus from water. In their procedure, 1- to 5-liter samples of surface-water are first clarified by filtration through an AP 20 MF fiberglass pre- filter. To the virus-contaminated water is then added 200 mg FeCl^ per liter of water. The water is adjusted to pH 6.0 to 6.5 and stirred for 60 tainutes. An in situ flocculation occurs with the formation of Fe(OH)^. The floe is then entrapped on a DWAP Millipore absorbent filter pad and subsequently the virus is eluted with 3% beef extract (Difco), pH 8 by passing a 10-ml volume of eluent through the ferric hydroxide layer on the filter pad. Two additional elution steps are conducted using 5 ml of eluent each. In order to examine several 5-liter samples at one time, all the eluates collect- ed from the individual FeCl^ flocculation procedure are pooled. The eluate pools are then further concentrated by the aqueous polymer two-phase separation system (a discussion of this procedure appears later in this paper). The results of the combined methods have shown virus concentrations of 400-fold with virus recoveries of 25 to 30 percent. According to these researchers, the advantages of the combined methods are vested in the fact that (i) eluates of five, 5-liter samples can be pooled and then examined for virus ------- -29 following the aqueous polymer two-phase separation system concen- tration, and (ii) surface-waters do not require additional filtra- tion to eliminate bacterial contamination. The major limitation of the combined-methods technique is related to sample size; i»e.} only 25 liters can be conveniently handled at one time. England (1970) reported that the use of protamine sulfate (salmine) flocculation followed by filtration through a Millipore prefilter pad facilitated the recovery of reoviruses and adeno- viruses from sewage effluents but was of little value for recover- ing most enteroviruses (polioviruses, echoviruses, and coxsackie- viruses) . This investigator indicated that concentration factors up to 250-fold were readily achieved by the technique. Virus re- coveries have ranged from 80 to 100% with reovirus and adenovirus (England ,1970b)~ The concentration of viruses by adsorption to insoluble cross- linked copolymers of maleic anhydride (polyelectrolytes) was de- scribed by Johnson, et at, (1967). They reported that polymers based on divinyl benzene-crosslinked styrene/maleic anhydride copolymer can adsorb 100% of tobacco mosaic virus and more than 99.99% of poliovirus from aqueous suspensions. Virus was eluted from the polymer-virus complex by the use of 1 M sodium chloride with a 52% efficiency. Wallis, et at, (1969) concentrated viruses from sewage by adsorption onto an insoluble crosslinked copolymer of isobutylene ------- -30 maleic anhydride designated as PE60 (produced by the Monsanto Company, St. Louis, Mo.). In their initial studies, a batch- technique was developed. Briefly, the batch-technique consisted of adding washed PE60 (400 mg per gallon) to clarified sewage. The sewage was clarified by filtration through an MF fiberglass prefilter pad in order to remove particulate matter. To enhance virus adsorption, the acidity of the sewage filtrate was adjusted to pH 5.0 to 6.0. Virus was added and the mixture was stirred for 1 hour at 25° C. The polyelectrolyte-virus suspension was then filtered through a 47-mm fiberglass prefilter pad. The virus-laden polyelectrolyte was recovered from the filter pad with the aid of a spatula. Virus was eluted from the polyelectrolyte with 10% fetal calf serum, pH 8.0 or 9.0. In laboratory experiments, the virus recovery efficiency was 93 percent. The insoluble poly- electrolyte (PE60) virus-adsorption technique was also applied to field samples of raw sewage from Houston, Texas during April and May of 1968. In April, a total of 1,461 virus isolants were re- covered. In May, a total of 205 isolants were recovered. In this limited field study, the polyelectrolyte-virus-adsorption batch- method was compared in parallel with the membrane-adsorption and the aluminum hydroxide-adsorption methods for detecting vi/tuses in the raw sewage. The authors concluded that the difference in virus isolation frequencies among the three methods was not always ------- -31 statistically significant. However, the polyelectrolyte- adsorption method consistently gave the highest isolation rates and was considered the preferable method because of economy and the shortened time involved for processing the sewage samples. In the same report, Wallis et al.j (1969) described a modified thin-layer PE60 procedure in which the PE60 was "sandwiched" between two fiberglass prefilter pads. This "sandwich" modification prevented polyelectrolyte displacement during the filtration process. The procedure included a double- adsorption procedure in which eluted virus from the PE60 adsorbent was readsorbed onto PE52 (crosslinked copolymer of ethylene maleic anhydride). Virus was eluted from the PE52 adsorbent with physio- logical saline. The double-adsorption-elution procedure resulted in reducing a 1-gallon volume of sewage into a final volume of 3 ml with efficient recovery of virus. Grinstein, et at. (1970) applied the polyelectrolyte-adsorption method (batch-technique) to sewage and to a river stream receiving sewage effluents in Houston, Texas during July and August of 1968. Virus was isolated 5 miles downstream from the nearest sewage effluent outlet. Overall, during two months of sampling 76, 1-gallon samples were collected which yielded 12,855 virus isolants. The average number of isolants per gallon sampled varied from 45 to 286 PFU. The authors concluded that with the polyelectrolyte- adsorption method, it was now possible to monitor virus in natural ------- -32 waters more effectively. The application of the polyelectrolyte-adsorption technique to very large volumes of water up to 100-gallon quantities or greater has been studied by Wallis, et aZ. ( ). They used the "sandwich" or thin-layer filtration system. Briefly, the thin- layer of polyelectrolyte was prepared as follows: (i) an AP 20 MF fiberglass prefilter pad was placed on a filter-screen support and 800 mg of PE60 suspended in 200 to 300 ml of distilled-water were filtered onto the pad when the 90-mm Millipore filtration unit were used; 10 grams of PE60 was used for the 293-mm filtration unit, (ii) the polyelectrolyte thin-layer was examined for surface-covering integrity and then a second AP 20 MF fiberglass prefilter pad was placed on top of the polyelectrolyte layer to form the "sandwich". By the use of the thin-layer filtration system (90-mm filtration unit), 25, 50, 75, and 100-gallon quantities of virus-contaminated tap-water were subjected to virus recovery experiments. The results indicated virus recoveries ranging from 65 to 80 percent. The method was also applied to a virus-seeded 17,000-gallon swimming pool. By filtering 300 gallons (1135 liters) of the pool-water through the thin-layer polyelectrolyte (293-mm filtration unit) an efficiency of about 40% virus recovery was observed. Jakubowski and Hoff (personal communication^ 1970) are currently evaluating the polyelectrolyte-adsorption technique for concentrating ------- -33 and detecting viruses in tap-water and estuarine-water (salinity, 27 °/oo). By use of the thin-layer filtration system (142-mm filtration unit), 10-liter quantities of either tap-water or estuarine-water were subjected to virus recovery experiments. The filter load was either 1 or 2 grams of polyelectrolyte. In the tap-water experiments, the results indicated virus recoveries of 58 and 74% for the 1-gram and 2-gram filter loads, respectively. In the estuarine-water experiments, the results indicated virus recoveries of 12 and 17% for the 1-gram and 2-gram filter loads, respectively. These researchers also tested the polyelectrolyte- adsorption concentration system using naturally occurring virus present in feces obtained from vaccinated infants. The adsorption and elution results were similar to those obtained with cell culture-grown virus. Kalter, (personal communication3 1970) is also currently investigating the polyelectrolyte-adsorption method for concentrating viruses from water. By the use of PE60 as the copolymer, he has observed virus recoveries of 60% from 1-liter quantities of water. Berg (1970) has carried out experiments with the polyelectrolyte' adsorption "sandwich" technique using PE60. By the use of 1-liter volumes of distilled-water seeded with small amounts of virus (75 to 105 PFU/liter), relatively poor virus recoveries resulted. For example, recoveries observed with poliovirus type 1 were 51 to 53 ------- -34 percent. With echovirus type 7, recoveries ranged from 25 to 30% and with reovirus type 1, 14 to 31 percent. Application of the technique to 50-gallon (189-liters) samples of river-water (field trial) was considered encouraging, however, since as much as 19 PFU of virus have been recovered from samples taken long distances from outfalls along a fast-flowing river during the winter months. Berg concluded by stating that despite its low and erratic efficiency, the technique appears to be the most sensitive presently available for large volume water studies. England (personal aomimmvcation, 1970b) has evaluated the polyelectrolyte-adsorption method (modified after Walliset a!., 1969) for concentrating and isolating viruses from raw sewage and sewage effluents. With sample volumes of 2-liter size, concentration factors up to 500-fold have been achieved. In laboratory-controlled experiments, recovery of enterovirus apd reovirus was 90 to 100% when the input-virus multiplicities were high; e.g., 1 to 10,000 PFU per ml. When the input-virus multi- plicity was low; e.g., <0.2 PFU per ml, recovery was very poor except for poliovirus. To summarize, the use of precipitable salts, iron oxide, and polyelectrolytes for concentrating and detecting viruses from water holds good promise as being an acceptable method. Experimentally, virus recoveries have ranged from 12 to 100% depending on the ------- adsorbent used, the test virus, and the type of water being examined. Experimentally, volumes of water from 400 ml to 300 gallons have been evaluated for virus concentration with favorable results. Some typical results are shown in Tables A and 5. Appli- cation of these virus-adsorption techniques to the real world situation has not been extensive. By use of the aluminum hydroxide floe technique and the polyelectrolyte-adsorption technique, however good results were observed with l-.gallon (3.78 liters) field samples of sewage and sewage effluents (Wallis, et at, 1969; Grinstein, et at, 1970). The use of iron oxide in field studies has not been studied. The advantages of using selected adsorbents for concentra- ting and detecting viruses from water are related to their simplicity speed, and economy. The major disadvantages of these methods are related to the quantity and types of water to be examined for virus. For example, the aluminum hydroxide floe technique is confined to samples of about 2 gallons; the iron oxide and polyelectrolyte- adsorption techniques are adversely affected by high turbidity which clogs the systems. Cost considerations would be considered reason- ably low except for the stainless-steel filtration assembly required to support the fiberglass prefilter pads and adsorbent. These filtration assemblies approach $900.00 for the 293-mm size unit. ------- TABLE 4 RECOVERY OF virus from water by adsorption to precipitable salts and iron OXIDE Percent of virt Reference Wallis and Melnick, 1966 England, 1970 et altJ 1968 Rao, 1970 Jakubowski and Hoff, 1970 Wetcalf, 1970 SchJffer and Borneff, 1970 Volume water Adsorbent used 1 liter Sewage effluent Al(OH) j 80 - X00 400 ml Sewage effluent Al(OH) 87 - 90 500 ml £p —w9lt G t Iron oxide - 100 150 liters Tap-water Iron oxide England, 1970 Iron oxide Iron oxide Iron oxide FeClj protamine sulfate Estuarine-water ¦jap—water Tap-water Surface-water Sewage effl^ent 10 liters 10 liters 1-10 liters 5 liters 1 liter 22 - 37 40 - 64 80 - 45 - 100 80 - 100 ------- TABLE 5 RECOVERY OF VIRUS FROM WATER BY ADSORPTION TO POLYELECTROLYTE Reference Type of water Volume of water Percent of virus recovered Johnson, et at., 1967 Distilled-water 5 ml - 52 Wallis, et alm> 1969 Sewage effluent 1 gallon - 93 England, 1970 Sewage effluent 2 liters 90 - 100 Jakubowski and Hoff, 1970 Tap-water Es tuarine-water 10 liters 10 liters 58 - 74 12 - 17 Kalter, 1970 Tap-water 1 liter - 60 Berg, 1970 Distilled-water 1 liter 14 - 53 Wallis, et at( ) Tap-water Swimming pool-water 100 gallons 300 gallons 65 - 80 - 40 ------- -36 Aqueous polymer two-phase separation method. The mechanism of polymer two-phase separation is liquid- liquid partitioning; a phenomenon closely related to adsorption. Partitioning occurs as a result of differences in particle surface properties and their distribution between two liquid phases while adsorption occurs as a result of differences in particle surface properties and their distribution between a solid phase and a liquid phase (Schwerdt, 1965). Basically, the aqueous polymer two-phase separation system consists of dissolving two different polymers such as dextran and methylcellulose or dextran and polyethylene glycol in water under specified conditions of salt, pH, and polymer concentrations. Following a holding period, usually 18 to 24 hours in the cold, two phases are produced. One phase, the bottom dextran phase, is small in volume and should contain the concentrated virus. The partitioning of particles in aqueous polymer two-phase separation systems of dextran sulfate and polyethylene glycol was reported by Albertsson (1958), The application of Albertsson's two-phase separation system for purification and concentration of viruses was described by Philipson, et at, (1960). The concentration of enteric viruses from water by a single- step and a two-step aqueous polymer two-phase separation system was ------- -37 described by Shuval, et at. (1967). In their system, the single- step, two-phase procedure was conducted as follows: (i) 640 ml of test sample was added to a mixture containing 0.2% (w/w) sodium dextran sulfate 2000 (A. B. Pharmacia, Sweden), 6.45% (w/w) poly- ethylene glycol (carbowax 4000), and 0,3 M sodium chloride; (ii) the mixture was shaken, transferred to a separatory funnel and held overnight at 4° C; (iii) the small bottom phase was then drained off together with the interphase; -(iv) potassium chloride was added to a final concentration of 1 molar to the drained phases to precip- itate the dextran sulfate; and (v) the mixture was then centrifuged at 2000 rpm for 5 to 10 minutes and the supernatant fluid assayed for virus. In the two-step, two phase procedure, sodium chloride was added to a final concentration of 1 molar to the bottom phase as obtained in the single-step procedure. The dextran sulfate was not precipitated with KC1. Following an additional holding period of 18 hours at 4° C, a new two-phase system developed; virus being concentrated in the small top phase. In Shuval and associates preliminary experimental studies, poliovirus was concentrated 52- to 200-fold by the single-step procedure with virus recovery effici- encies ranging from 37 to 98 percent. The two-step, two-phase procedure yielded a 274-fold virus concentration. The researchers indicated that virus multiplicities as low as 0.066 PFU/ml were detected by the two-phase system. They further speculated that ------- -38 10 PFU/liter or less may be detectable by the method. Further development and refinement of the polymer two-phase separation system for concentrating enteroviruses in water was reported by Shuval, et a1a in 1969. In the refined system, the procedure was shown, experimentally, to achieve a median virus concentration of 520-fold with virus recoveries ranging from 35 to greater than 100% from water. As few as 1 to 2 PFU per liter of sample were detected about 85% of the time. The method was also evaluated by experimen- tally seeding sewage effluent with poliovirus (2,7 to 1418 PFU/ liter). The average physical concentration factor was 224, The virus recovery efficiency ranged from 62 to greater than 100 percent. The aqueous polymer two-phase separation technique was also tested in field trials by examining raw sewage, water from wells, springs, and streams, effluent from oxidation ponds, and effluent from a biofiltration plant. One-liter samples were collected. Viruses were detected in virtually all of the field samples of sewage with a maximum of 11,184 PFU/liter in raw sewage. Virus was also de- tected in 2 samples of drinking water from a shallow municipal well. The researchers concluded that the use of the aqueous polymer two-phase separation method for field surveys of sewage and water samples illustrated the value of the method for the routine monitoring of potable water supplies for virus contamination. The use of an aqueous polymer two-phase separation system for ------- -39 concentrating enteroviruses from sewage has also been reported by Lund and Hedstrom (1966). In their procedure, sodium dextran sulfate 2000 was employed as a 20% solution by weight while poly- ethylene glycol (carbowax 6000) was used as a 30% solution by weight. Samples collected from the field survey were handled as follows: (i) to 200 ml of sewaje were added 20 grams of 5 M NaCI, 58 grams of 30% polyethylene glycol, and 2.7 grams of 20% sodium dextran sulfate. The acidity of the mixture was then adjusted to pH 7.2. After'shaking, the sar.ple was held for 24 hours at 4° C before being assayed for virus. During the field survey, weekly samples were collected. From the end of June to December 1966, a total of 40 virus isolations '.:ere made. The samples processed by the aqueous polymer two-phase separation technique yielded 38 virus isolations while only 15 samples of untreated sewage samples were positive for virus. The researchers concluded that the method was efficient for concentrating virus from sex?age, Lund (.-personal cormunioabi 1970) has also applied the aqueous polymer two-phase separation system to water samples. Samples were collected both by the swab-method (polyethylene sponge) and by the dip-sample method. Swab samples were handled as follows: (i) swabs were exposed to water to be examined for 24 hours; (ii) 200 ml of fluid were squeezed from the swab; and (iii) the sample was then concentrated in the usual manner by adding sodium dextran ------- -40 sulfate, polyethylene glycol, and sodium chloride. Dip-samples were processed using 200-ml volumes. The physical concentration was 100-fold, Following the two-phase separation, the bottom phase and interphase were processed in parallel. Both samples were decontaminated by ether treatment and then inoculated directly into tissue culture. It was observed that with field samples a greater number of virus-positive samples were obtained by using the swab-method of sample collection. In laboratory- controlled experiments, virus recovery from water samples was observed to be 100 percent. Interestingly, Lund noted that with sludge samples no concentration was obtained with the aqueous polymer two-phase separation system. Limitations of the aqueous polymer two-phase separation tech- nique for detecting viruses in dilute aqueous suspensions'were re- ported by Grindrod and Cliver (1969), Using primary rhesus monkey kidney for virus quantification, 7 enteroviruses (poliovirus types 1, 2, 3; coxsackievirus A-9; coxsackievirus B-2 and B-3; and echovirus type 6) were subjected to concentration by the aqueous polymer two- phase separation technique. At a high-multiplicity of virus input 3 8 (1.4 x 10 to 6.5 x 10 PFU), all seven enteroviruses Rested were efficiently concentrated into the bottom dextran phase. At a low- multiplicity virus input (1 to 10 PFU), recovery of poliovirus type 1 was considered satisfactory while the recovery of coxsackievirus ------- -41 A-9 was unsatisfactory (recovery <11%). During the course of experimentation, it was observed the sodium dextran sulfate was somewhat inhibitory to coxsackievirus A-9, and extremely inhibitory to coxsackievirus B-2 and echovirus type 6. The researchers sug- gested that inhibition of the model viruses used in quantitative experiments might have escaped notice by other workers because the bottom dextran sulfate phase was diluted before titrating the viruses. The researchers summarized their results by stating that the aqueous polymer two-phase separation technique was found to be a significant aid to detection of all three polioviruses, coxsackie- virus B-3, and in the presence of fluid maintenance medium only, coxsackievirus A-9, The method, however, was considered worse than no treatment at all for detecting coxsackievirus B-2, and echovirus type 6. Cliver (personal oomrrwoxiaationj 1970) and in a recent pub- lication (Grindrod and Cliver, 1970) indicated that there should be a 50% probability of detecting virus in a sample containing 1 to 2 PFU per liter, depending on the virus type, of course, and if dextran rather than sodium dextran sulfate is used in the two-phase separation system. Recoveries of enteroviruses were observed to range from 59 to 164% when tested with dextran as compared to 0,001 to 100% with dextran sulfate. Liu, et at. ( ) have studied some of the parameters that influence the efficacy of the aqueous polymer two-phase separation ------- -42 system for concentrating and recovering viruses from water and seawater. They worked with poliovirus type 1, coxsackievirus A-9, echovirus type 11, adenovirus type 12, and influenza virus type A. They observed that sodium dextran sulfate 2000 and 500 were equally effective. Conversely, sodium dextran sulfate of smaller molecular size and DEAE-dextran were totally ineffective. The optimal poly- ethylene glycol as well as the sodium chloride concentration was found to vary depending on the virus being studied. They con- cluded that the4 use of the aqueous polymer two-phase separation technique for separating a mixture of viruses could be a very complex problem. Their results indicated that many facets of the technique remained to be investigated; e.g., (i) more virus types should be studied; and (ii) optimal concentrations of sodium dextran sulfate, polyethylene glycol, and sodium chloride for each virus type must be determined. Virus recovery from 2-liter volumes in laboratory-controlled studies has ranged from 57 to 100 percent. In a pilot field-survey study, Liu, at, ( ) observed that sample sizes of 20 liters were impractical because a preliminary volume-reduction step was required. This was done by hydroextrac- tion with polyethylene glycol. They routinely processed samples of 2-liter size by the aqueous polymer two-phase separation tech- nique without difficulty, Nupen (1970) has applied the aqueous polymer two-phase sepa- ration technique in field trials for^virus detection in waste-water. ------- -43 The field trials were undertaken in connection with the virus con- trol program for testing the Windhoek advanced waste-water recla- mation plant at Windhoek, South Africa. The technique as described by Shuval, et at. (1969) was employed with the exception of the following: (i) 2 grams sodium dextran sulfate 500 (A. B, Pharmacia, Sweden), (ii) 6.45 grams polyethylene glycol (carbowax 20,000), and (iii; 17.53 grams sodium chloride were added to each liter of water sample. Virus recovery from 1-li.ter volumes in laboratory-controlled studies was 40 percent. An evaluation of virus quantification from the aqueous polymer two-phase separation technique by the TCID^q assay method and by the plaque technique was also undertaken. In this regard, the TCID^q method was considered to be superior to the plaque method, experimentally, as determined by the coefficient of variation among replicate titrations. The experiment was conducted using poliovirus type 2 (P712) added to tap-water. During the testing of the Windhoek reclamation plant, com- posite water samples were prepared by collecting 500-ml samples from each sampling point every 6 hours for a period of 48 hours. The sampling points were: (i) settled sewage, (ii) humus tank effluent, (iii) maturation pond effluent, immediately before advanced treat- ment, (iv) Goreangab Dam effluent, and (v) the treated water. One- liter quantities of water were concentrated by the aqueous polymer two-phase separation technique. Virus was isolated from the settled ------- -44 sewage, the humus tank effluent and after 14 days' retention in the maturation pond effluent. No virus was detected after advanced waste-water treatment. It was noted that, in the field samples, a higher isolation rate was obtained by the TCID^q assay method when a comparison was made with the plaque assay technique. The researcher concluded by stating that the aqueous polymer two-phase separation technique proved effective in testing of the virus removal by the advanced waste-water treatment plant. To summarize, the use of the aqueous polymer two-phase separa- tion technique for concentrating and detecting viruses from water holds promise as being an acceptable method. Experimentally, virus recoveries have ranged from 35 to greater than 100 percent. Some typical results are shown in Table 6. The method is limited apparently to 1- or 2-liter volur.es of water. Application of the method to field samples has been carried out by a number of workers with favorable results (Shuval, 1969; Lund and Hedstrom, 1966; and Nupen, 1970). Indications are that virus multiplicities as low as 1 to 2 PFU per liter of water can be detected with about 85% reliability (Shuval, 1969). The major advantages of the technique are simplicity and economy. One major disadvantage of the method may be related to the inhibitory action of dextran sul/ate on certain enteroviruses (Grindrod and Cliver, 1969) However, Liu, et at, ( ) indicated that with KC1 precipitation, the dextran ------- TABLE 6 RECOVERY OF VIRUS FROM WATER BY THE AQUEOUS POLYMER TWO-PHASE SEPARATION METHOD Reference Type of water Volume of water Percent of virus recovered Shuval, et al,3 1967 Distilled-water 640 ml 37 - 98 Shuval, et al. 3 1969 Sewage effluent 1 liter 35 - 100 Lund, 1970 Sewage effluent 200 ml - 100 Cliver, 1970 Tap-water 1 liter 59 - 164 Nupen, 1970 Waste-water 1 liter - 40 Liu, et al, j ( ) Tap-water 2 liters 57 - 100 ------- -45 sulfate is effectively eliminated from the bottom phase. The small quantity of water that can be processed at a given time may be another disadvantage; i.e., if the method is applied to rela- tively nonpolluted or finished (drinking) waters. It would appear that the method was more effective with moderately or grossly pol- luted waters; with finished waters, the method may be equivocal. Soluble alginate filter technique. The mechanism of soluble alginate filters that permits the concentration of viruses has not been precisely proffered in the literature but undoubtedly involves a sophisticated virus entrap- ment and may even involve a cor.bination of retention and adsorption. Nevertheless, the unique feature of the alginate filter is related to its solubility in sodium citrate. And, for the most part, the alginate-citrate solution is not virus-inactivating nor cytotoxic. In the early work, filters were prepared by the individual investi- gator. This was done, for exar.ple, by placing a solution of 1% sodium alginate sol onto a piece of filter paper previously soaked with an electrolyte consisting of 0.5 M lanthanum nitrate [La(NC^y 6H2o3 and 0.5 M aluminum chloride (AlCl^I^O). Following filtra- tion, a 3.8% solution of sodium citrate is used to dissolve the alginate filter. Tissue culture or mice are then inoculated di- rectly with the alginate-citrate solution (G&rtner 1967). Alginate ------- -46 filters are now available commercially in the United States (Sartorius Membranes Division, Brinkman Instruments, Inc., Westburg, N.Y. 11590) and also in Germany (Sartorius). The efficiency of the laboratory-prepared alginate filters was reported by Gartner in 1967. Working with poliovirus as the model virus in filtration studies, Gartner presented evidence that the alginate filter completely retained the input-virus; no virus being found in the filtrate. The experimental recovery of polio- virus from dissolved filters ranged from 25 to 100% being somewhat influenced, perhaps, by the initial input-multiplicity of virus. Field trials were also undertaken by G&rtner in which untreated sewage and sewage effluents from purification plants were examined for virus content by use of the homemade alginate filters, A com- parison of the direct inoculation method to the soluble alginate filter technique yielded 55% and 87% virus-positive isolations, respectively. This indicated the superiority of the soluble alginate filter method over the direct inoculation method. Gartner (personal communication, 1970) has further indicated that 1-liter volumes of clean water can be filtered in a short time. With pol- luted (turbid) waters, however, the filtration flux is noticeably reduced, G'drtner (1970) recognized that the soluble alginate filter technique may not be the ultimate method but he feels that it is the best method presently available for concentrating and detecting ------- -47 viruses from water. Witt (1965) presented information that up to 10 liters of water could be filtered through the soluble alginate filter and then by dissolving the filter in 1 ml of isotonic citrate solution, a concentration of 10,000 to 1 could be achieved. In laboratory experiments using poliovirus tyje 3, he was able to detect as little as 0,001 LD^/ml, Mitt observed some difficulties in the beginning when the alginate method was applied to field samples of drinking water. With increasing volumes of water, he found a corresponding increased filtration flux that negated the filtra- tion process. This difficulty vas overcome in later field studies by combining the alginate filter with a membrane support filter. In these field studies, no viruses were isolated from the drinking water, Nupen (1970) has applied tr.e soluble alginate filter technique in field trials for virus detection from waste-water# The field trials were undertaken in connection with the virus control program for testing the Windhoek advanced waste-water reclamation plant at Windhoek, South Africa, By the use of sterile Sartorius-membrane filters (GmbH 50-trm) under 500 m Hg negative pressure, 1-liter volumes of waste-water were filtered through the alginate-membrane supported filter system. The virus-containing-alginate film was floated off the supporting membrane with 0.9% sodium chloride and then dissolved in 3 ml of 3,8% sodium citrate solution. During the ------- -48 field trials, virus was isolated from the settled sewage, the humus tank effluent and after 14 days1 retention in the maturation pond effluent. No virus was detected after advanced waste-water treat- ment. These results were the same as those observed with the aqueous polymer two-phase separation technique. A laboratory eval- uation of the soluble alginate filter technique by Nupen showed an average virus recovery of 40.05% and a coefficient of variation of 41.69 percent. According to the researcher, this further indi- cated that the alginate method was comparable to, and of equal efficiency as, the aqueous polymer two-phase separation technique. However, Nupen (personal oo^runioation^ 1970) also indicated that the soluble alginate filter technique was impractical time-wise on turbid water samples because of clogging problems. Poynter, (personal convr.cnioation^ 1970) has used the soluble alginate filter technique over the past six years and is currently using the method for isolation of viruses from river waters in England, Alginate filters were at one time prepared by hand at Poynter's Laboratory but now are obtained commercially from Sartorius (Germany). The cornmercial filters have been reinforced so that pressures of up to 700 mm Hg may be used. According to Poynter, the alginate filter technique provides the simplest and yet the most effective method for examining environmental waters for viruses. In practice, turbid waters are prefiltered through ------- -49 Oxoid cellulose acetate membrane filters (47-mm, 0.45 ym porosity) pretreated with broth (10 ml of Hartleyfs digest broth or a protein-containing fluid such as serum) to prevent virus adsorp- tion. The crystal clear water is then filtered through the alginate filter. Up to 2 liters per alginate filter may be pro- cessed at one time. Filtration is done in a cold-room at 5° C. Following filtration, the alginate filter is detached from the reinforcing backing-filter piece and dissolved in isotonic sodium citrate (3.8%). Viruses present in 1 to 2 liters of water are thus concentrated into a 1-ml volume. After addition of antibiotics, the alginate-citrate solution is inoculated directly onto tissue culture. Raw water samples of 19-liter size have also been pro- cessed with a special pressure vessel. Rupture of the alginate filter with the larger volume has not been observed. During the past several years of field experience, Poynter has observed that enteroviruses are regularly recovered from the River Thames and the River Lee at counts of 0.5 to 10 PFU/liter. In laboratory studies, Poynter (1970) indicated that virus recoveries have ranged from 60 to 70% to over 100 percent. Viruses have never been found in the filtrates following filtration. To summarize, the use of the soluble alginate filter technique in laboratory experiments and in field trials indicates that the soluble alginate filter method holds good promise for being an ------- ¦50 adequate and acceptable method for concentrating and detecting viruses from water. Experimentally, virus recoveries ranging from 25 to 100% have been reported. Some typical results are shown in Table 7. Volumes of water normally subjected to virus assessment have beet» 1 or 2 liters. However, 19-liter volumes have also been processed successfully. Application of the soluble alginate filter technique to the real world situation has been carried out for over six years by British investigators (Poynter, 1970) and by investigators in Germany (GHrtner, 1970) and South Africa (Nupen, 1970) with favorable recovery results. The advantages of the soluble alginate filter technique for concentrating and detecting viruses in water are related to its simplicity, speed, and econ- omy with particular emphasis being placed on the solubility feature of the filter material. The major disadvantage of the method mani- fests itself when turbid waters are subjected to virus examination because the filters clog rather easily. Consequently, waters con- taining suspended particulate matter must be prefiltered before filtration by the alginate filter, Continuous-floi) v.Itracentrifugaticn method. The mechanism of this method is related to the sedimentation characteristics of virus particles (virions) subjected to a cen- trifugal force. Today, commercial ultracentrifuges have capacities ------- TABLE 7 RECOVERY OF VIRUS FROM WATER BY THE SOLUBLE ALGINATE FILTER TECHNIQUE Percent of Volume of virus Reference Type of water water recovered Gartner, 1967 Drinking-water 10 liters 25 - 100 G&rtner, 1970 Surface-water 1 liter - 100 Nupen, 1970 Waste-water 1 liter - 40 Poynter, 1970 River-water 2 liters 60 - 70 ------- -51 of 100- to 1000-ml and are capable of speeds yielding average centrifugal fields of 40,000 to 120,000 times gravity. Small viruses, the size and density of poliovirus virions can be complete- ly sedimented within 1 to 1.5 hours from an aqueous suspension by a centrifugal field of 120,000 times gravity (Schwerdt, 1S65). Depending upon the size, shape, and density of virus particles, the centrifugal force, viscosity, and density of suspending medium, it is possible to fractionate classes of particles by either rate- zonal or isopycnic-zonal centrifugation. In rate-zonal centrifu- gation, particles are separated on the basis of differences in sedimentation rate in a density gradient. The density gradient provides gravitational stability to the system. In isopycnic- zonal centrifugation, particles having the same sedimentation rate are separated on the basis of different buoyant densities. The adaptation of isopycnic-zonal centrifugation to concentration of viruses from water relies on the fact that this procedure can be carried out as continuous-flow ultracentrifugation whereby a con- tinuous stream of water is flowed over a density gradient in a special rotor (Anderson, et al. 1967). The application of routine ultracentrifugation for concentra- tion and detection of enteroviurses in dilute suspension was described by Oliver and Yeatman (1965), By use of a Spinco Model L Preparative Ultracentrifuge with both the number 30 and 50 rotors, ------- -52 quantitative recovery of poliovirus type 1 and coxsackievirus B-2 was achieved. Samples of less than 10 ml can be concentrated in 120 minutes in the number 50 rotor. The researchers concluded that there was a 50% probability of detecting enterovirus by means of the number 50 rotor when the initial virus multiplicity was as low as 0.12 PFU/ml. The same findings were proffered with the number 30 rotor when the initial virus multiplicity was as low as 0.025 PFU/ml. Cliver (personal eotrmuni.oationt 1970) indi- cated that by means of the number 50,1 rotor, 840 ml of sample t can be processed per day. Concentration of virus has ranged from 35- to 70-fold. Experimentally, virus recoveries have ranged from an average of 60 to 70% with a high of 118 percent. The method has not been applied to field samples. The application of continuous-flow ultracentrifugation for concentrating viruses in water was presented by Anderson in 1965 at a national symposium (Anderson, et at. 1967), A high perfor- mance continuous-flow centrifuge was described which removed over 95% of suspended poliovirus at a flow rate of 2 to 3 liters per hour. The sedimented virus was pelleted directly onto the wall of the rotor and removed by resuspension. It was noted by Anderson that certain types of virus, however, were inactivated after pel- leting. This difficulty was overcome by trapping virus particles isopycnically in stationary density gradients. The experimental ------- -53 systems built were successfully tested with adenovirus type 2 and respiratory syncytial virus. According to Anderson (1970) continuous-flow centrifuges for large-scale isolation of viruses from dilute suspensions are being developed. For example, the K-II zonal ultracentrifuge at flow rates above 25 liters per hour has yielded influenza virus recoveries of 80 to 90 percent. Higher-capacity centrifuges are also being designed. These cen- trifuges not only concentrate the virus but band it isopycnically at the same tiifle. To summarize, the use of preparative ultracentrifugation for concentrating and detecting viruses from waters of the real world does not hold too much promise as a routine method. The major disadvantages of the method are: (i) the ultracentrifuges are too costly to buy and maintain; and (ii) processing large volumes of water would be prohibitively time-consuming. The application of ultracentrifugation techniques to virus-in-water problems of the real world may involve their use as a secondary-procedure for final virus concentration prior to viral assessment in tissue culture. Preparative ultracentrifugation could be particularly useful for concentrating viruses iij eluates from virus adsorption- elution methods. Continuous-flow ultracentrifugation, at the present time, must be considered as developmental, the application, of which remains to be determined. ------- -54 Forced-flew electrophoresis and electro-osmosis. The mechanism of electrophoresis and electro-osmosis is related principally to the amphoteric nature of viruses in that viruses exhibit mobility in a predictable direction at a given pH under a direct current electrical field. In forced-flow electrophoresis, electrophoretic transport brings about selective adsorption of viruses onto dialyzing membranes. The electrophoretic cells used in this method are essentially filtering devices. The apparatus which' consists of semipermeable dialyzing membranes and a microporous filter separated by suitable plastic spacers has been described in detail by Bier, et at• (1967). In electro- osmosis, water diffuses from one side of a membrane to the other under the influence of a difference of electrical potential. For example, when water is confined in a capillary tube, adsorption of hydroxy1 ions (0H~) imparts a negative charge to the walls of the tube. Adjoining the hydroxyl ions is a layer of hydrogen ions (H+) in equal number producing an electrical double-layer. If a difference of electrical potential exists between the two ends of the tube, water migrates toward the negative electrode by a pro- cess referred to as electroendosmos'is. The presence otf anions such as viruses (suspended at a pH above their isoelectric point) are firmly adsorbed to the walls of the tube during the process. ------- -55 The application of forced-flow electrophoresis to concentra- tion and detection of viruses in water was reported by Bier, et at, (1967). When using bacteriophage as the model virus, and flow rates of 60 to 240 ml per hour, quantitative recovery of virus was achieved. Concentration of bacteriophage was observed to be approximately 100-fold. According to the researchers, forced-flox^ electrophoresis has several advantages: (i) large volumes of water can be processed in a short time; (ii) the proce- dure and equipment are simple; and (iii) bacterial contamination can be eliminated from the virus suspension. Interestingly, it was noted in forced-flow electrophoresis that the application of electric current prevents clogging of the microporous membrane filter and decreases adsorption of virus onto the filter matrix. The method has not been applied to field samples. According to Bier (personal communiaation3 1970), the sensitivity of the tech- nique, as determined experimentally, is of the order of a few PFU per ml while recovery of virus is near 100 percent. Complete retention of all the virus within the electrophoretic cell has been obtained, McHale, et at, (1970) described the concentration of virus from water by forced-flow electrophoresis and electro-osmosis. They used an electrophoretic filter/concentrator modified after Bier. When using poliovirus as the model virus and flovr rates of ------- -56 approximately 300 ml per hour, virus was concentrated nearly 3-fold by forced-flaw electrophoresis. By the use of electro- osmosis, concentration of virus was 5-fold; water being removed at a rate of 0,8 ml per hour per cm of membrane area. Under the conditions of experimentation, the investigators concluded that forced-flow electrophoresis and electro-osmosis were more rapid and gentle than other methods for concentration of virus. To summarize, the use of forced-flow electrophoresis and electro-osmosis techniques for concentrating viruses from water t appears to be developmental but may have some application. Con- centration of virus from water has ranged from 3- to 100-fold. The small volumes of water that can be processed in a reasonable time period, however, would tend to malce these methods of little value as primary virus concentration techniques for very large volumes of water. Their application as secondary virus concentra- tion procedures may be worth considering. Neither technique has been applied to the real world situation. Other methods, Cliver (1967) described the use of polyethylene glycol in a hydroeKtraction method for concentrating virus from water. In this method, a 100-ml sample is placed into a dialysis tube which is surrounded by 100 grams of polyethylene glycol {carbowax 20,000) dissolved in 100 ml of wat-ir. Concentration Is allowed to ------- -57 proceed at room temperature. After 2 to 3 hours, the volume of the 100-ml suspension was reduced to 1 ml, achieving, of course, a 100-fold physical concentration. Virus recoveries have ranged from 10 to 30% (Oliver, personal oommunication, 1970), Indications are that there is a 50% probability of detecting virus in liter- quantities of water if the contamination level is at least 10 PFU per liter. Hoff, et alB (1967) designed and evaluated a flow-through gauze sampler-device for concentration of viruses from water. The sampler-device was tested during some field trials on a stream remote from human habitation. In the field studies, 1 gallon per minute was pumped through the sampler-device for 7 days (1440 gallons per day). No virus was detected during the course of the field trials. Experimental laboratory studies indicated virus recoveries ranging from 0.6 to 3.5% depending upon whether the water sample was clear or turbid; higher virus recoveries being observed when the water was turbid. Hoff (personal QormunioaHont 1970) indicated that the main advantage offered by the flow-through gauze sampler-device was that large volumes of natural waters could be sampled over extended periods of time (1440 gallons per 24 hours). Nevertheless, the efficiency and quantitative recovery of virus from water was discouragingly low in experimental studies. Liu, et at. (1970) reported on a preliminary study that was carried out on the flow-through gauze sampler-device designed by ------- -58 Hoff and associates for recovering virus from tap-water and seawater. The amount of virus (poliovirus) recovered by the sampler-device was 2% when tap-water was used. In seawater, virus recovery ranged from 15 to 19 percent. The addition of 3% sodium chloride to the tap-water increased the virus recovered to 47 percent. Volumes of water up to 90 gallons were tested by the sampler-device. The flow rate of the sampler-device was 1-gallon per minute. The virus con- centration factor achieved when seawater was used was 100-fold; while in tap-water the virus concentration factor achieved was about 10-fold, ' The researchers concluded that the flow-through gauze sampler-device should be further developed and evaluated. GENERAL DISCUSSION A number of methods adapted to concentrating and isolating viruses from water have been described. Several of the methods hold good promise as being acceptable for routine use in the real world situation. For example, the membrane-adsorption technique, adsorption to iron oxide or polyelectrolytes, aqueous polymer two-phase separation, and the soluble alginate filter technique all have shown good-to-excellent virus recovery efficiencies under laboratory-controlled conditions. Under field conditions, common ------- -59 deficiencies have been observed with some of these methods when the water was turbid (muddy); a common feature of many surface- waters. For example, the membrane-adsorption technique, the iron oxide or polyelectrolyte "sandwich" technique and also the soluble alginate filter technique all fall short for examining moderately turbid water because of clogging problems. This difficulty can be overcome to some extent by clarification of turbid water samples by prefiltration before applying the particular virus con- centration method. The need to prefilter turbid waters places an ill-defined limitation, however, on a particular method since pre- filtration may result in significant losses of virus in the water sample. If low-multiplicities of virus occur in the water sample their presence may be undetected. It would appear that the aqueous polymer two-phase separation technique is best suited for quantita- tively detecting and isolating viruses from moderately turbid waters. The major disadvantage of this method is related to the small volume of water that can be processed at a given time; e.g., 1- to 2-liter samples. Therefore, the waters must be moderately or grossly polluted with fecal wastes in order to achieve a high number of successes (virus isolations). The major advantages of the aqueous polymer two-phase separation technique are simplicity and economy. In clean water or finished waters, the problem of clogging would be nil but the expected occurrence of viruses at extremely ------- -60 low-multiplicities would demand that very large volumes of water of at least 100 gallons be processed. It would appear that the virus-adsorption and/or virus-retention procedures; i.e., virus adsorption to microporous membranes, iron oxide, and polyelectro- lytes; and virus retention by soluble alginate and possibly the gauze flow-through sampler-device are best suited for quantita- tively detecting and isolating viruses from clean or finished waters. In addition to the very large volumes of water that can be pro- cessed by the virus-adsorption and/or virus retention procedures, they have the common advantage- of speed, simplicity, and economy. These features will be particularly appealing when a technique is eventually selected as a candidate for routine use. Initial costs, however, may seem relatively high when considering the cost of the stainless-steel filtration assemblies or holders. These can often approach $900.00 for the larger size holders. The shortcomings of contir.uous-flow ultracentrifugation for routine use for quantitatively detecting and isolating viruses from waters are several-fold, the least of which is very high cost. The application of continuous-flow ultracentrifugation to the real world problems of viruses-in-vater would appear to be impractical. Likewise, forced-flow electrophoresis and electro-osmosis would appear to be primarily research tools rather than having any practical application for quantitatively detecting and isolating viruses from naturally contaminated waters. ------- Acknowledgements We thank Abner C. Jones III for preparing the photographic material. We also thank Vida H. Hartfield for typing the manus ------- -61 REFERENCES Anderson, N. G,, Cline, G. B., Harris, N. W., and Green, J. G. Q967) "Isolation of Viral Particles from Large Fluid Volumes" in Transmission of Viruses by the Water Routes G. Berg, ed. Interscience Publishers, New York. Anderson, N. G. (1970) Public Health Aspects of Viruses in Watera N. A, Clarke, ed, Cincinnati, Ohio, 1, p, 1. Albertsson, P. A. (1958) "Particle Fractionation in Liquid Two Phase Systems," Biochem, Biophys, Acta, 27, pp. 378- . Berg, G., ed. (1967) Transmission of Viruses by the Water Route, Interscience Publishers, New York, Berg, G, (1971) "An Integrated Approach to the Problem of Viruses in Water," Proceedings National Specialty Conference on Disin- fection, Amherst, Mass, July 8-10, 1970, Bier, M., Bruckner, G, C., Cooper, F. C., and Roy, H. E. (1967), "Concentration of Bacteriophage by Electrophoresis," in Transmission of Viruses by the Water Route, G. Berg, ed. Interscience Publishers, New York, Bier, M, (1970) Personal conmunication. Veterans Administration Hospital, Tucson, Arizona 85713, Bloom, H. H., Mack, W, N,, Krueger, B. J., and Mailman, W. L. (1959) "Identification of Enteroviruses in Sewage," Journal of Infectious Diseases, 105, pp. 61-68. Borneff, J. (1970) Public Health Aspects of Viruses in Watert N, A. Clarke, ed. Cincinnati, Ohio, 1, p. 1, Chang, S. L., Berg, G., Busch, K, A., Stevenson, R, E,, and Clarke, N. A. (1958) "Application of the "Most Probable Number" Method for Estimating Concentrations of Animal Viruses by the Tissue Culture Technic," Virologyt 6, pp, 27-42, Chin, T, D. Y,, Mosley, W. H., Robinson, S., and Gravelle, C, R„ (1967) "Detection of Enteric Viruses in Sewage and ty&ter. Relative Sensitivity of the Method," in Transmission of Viruses by the Water Route, G, Berg, Ed, Interscience Publishers, New York, ------- -62 Cliver, D. 0. (1965) "Factors in the Merribrane Filtration of Enteroviruses," Applied Microbiologyt 13:3, pp. 417-425. Cliver, D. 0., and Yeatman, J, (1965) "Ultracentrifugation in the Concentration and Detection of Enteroviruses," Applied Microbiology, 13:3, pp. 387-392, Cliver, D. 0. (1967) "Detection of Enteric Viruses by Concentration with Polyethylene Glycol," in Transmission of Viruses by the Water Routet G. Berg, ed. Interscience Publishers, New York. Cliver, D. 0. (1968) "Virus Interactions with Membrane Filters," Biotechnology and Bioengineeringj X pp. 877-889. Cliver, D. 0. (1970) Personal communication. Food Research Institute, 2115 Herrick Drive, The University of Wisconsin, Madison, Wisconsin 53706, U.S.A. "Engineering Evaluation of Virus Hazard in Water," (1970) Committee on Environmental Quality Management, Berger, B, B. (Chmn), Journal of the Sanitary Engineering Division, ASCE, 96, pp. 111-161. England, B., Leach, R. E,, Adame, B., and Shiosaki, R. (1967) "Virologic Assessment of Sewage Treatment at Santee, California," in Transmission of Viuvses by the Water Route3 G. Berg, ed, Interscience Publishers, New York. England, B. (1970a) "Protamine Sulfate Precipitation of Reovirus and Adenovirus for Their Assay in Sewage and Effluents," Bacteriology ,"Proceedings 1970, p. 194. England, B. (1970b). Personal communication. Department of Public Health, County of San Diego, 1600 Pacific Highway, San Diego, California 92101. GSrtner, H. (1967) "Retention and Recovery of Polioviruses on a Soluble Ultrafilter," in Transmission of Viruses by the Water Route, G. Berg, ed. Interscience Publishers, New York, GS.Tttvert H. (1970) Personal cormunication, Direktor des Hygiene- Instituts der Universitat, University of Kiel, 23 Kiel, West Germany, Grabow, W. 0, K. (1968) "The Virology of Waste Water Treatment," (Review paper). Water Research^ Pergamon Press, 2, pp. 675-701, ------- -63 Grinstein, S., Melnick, J. L., and Wallis, C. (1970) "Virus Isolations from Sewage and from a Stream Receiving Effluents of Sewage Treatment Plants," Bulletin World Health Organization, 42, pp. 291-296. Grindrod, J., and Cliver, D. 0. (1969) "Limitations of the Polymer Two-Phase System for Detection of Viruses," Archil) fUr die Gesamte Virus forschung, 28, pp. 337-347. Grindrod, J., and Cliver, D. 0. (1970) "A Polymer Two Phase System Adapted to Virus Detection," Arohiv fiXr die Gesamte Virusforsohung 31. pp. 365-3720 Hamblet, F. E., Hill, W. F., Jr., and Akin, E. W. (1967) "Effect of Plaque Assay Diluent Upon Enumeration of Poliovirus Type 1. Applied Microbiology3 15:1, p. 208. Hill, W. F., Jr., Akin, E. W., Benton, W. H., and Metcalf, T. G. (_ ) "Virus in Water. Ill Concentration of Poliovirus from 100-Gallon Quantities of Water by Millitube MF Cartridge Filter Adsorption," (to be published)» Hoff, J. C., Lee, R. D., and Becker, R. C. (1967) "Evaluation of a Method for Concentration of Microorganisms in Water," American Public Health. Association Proceedings. Hoff, J. C. (1970) Personal communication. Northwestern Water Hygiene Laboratory, Route 4, Box 4519, Gig Harbor, Washington 98335. Jakubowski, W., and Hoff, J. C. (1970) Personal corrmunication. Northwestern Water Hygiene Laboratory, Route 4, Box 4519, Gig Harbor, Washington 98335. Johnson, J. H., Fields, J. E., and Darlington, W. A. (1967) "Removing Viruses from Water by PolyelectrolytesMature (London), 213, pp. 655-657. Joseph, J. M. (1970) Personal communication. Department of Health and Mental Hygiene, Bureau of Laboratories, 16 E. 23rd Street, Baltimore, Maryland 21218. Kalter, S. S. (1970) Personal comunication. Southwest Foundation for Research and Education, P.O. Box 2817, 10,000 West Commerce Street, San Antonio, Texas 78228. ------- -64 Kramer, H. P. (1965) "Research Needs for Drinking Water," Journal American Water Works Association3 57:1, pp. 3-9. Larson, S, 0., and Reinicke, V. (1965) "Some Statistical Problems in Relation to the Use of the Poisson Distribution in Virus Plaque Assays," Acta Path, et Microbiology, Scandinav. 65, pp. 84-92. Liu, 0. C., Brashear, D. A., Seraichekas, H. R., Barnick, J. A., and Metcalf, T. G. (1970) "Virus in Water. I. A preliminary Study on a Flow-Through Gauze Sampler for Recovering Virus from Water," (to be published), Liu, 0. C., Seraichekas, H. R., Brashear, D. A., Cipolla, R. J., Barnick, J. A., Carey, P. F., and Metcalf, T. G. ( ) "Virus in Water. II. Concentration of Viruses in Water by Two-Phase Polymer Method," (to be published), Lund, E., and Hedstrom, C. E. (1966) "The Use of an Aqueous Polymer Phase System for Enterovirus Isolations from Sewage," American Journal of Epidemiology3 84:3, pp. 287-291. Lund, E., and Hedstrom, C. E. (1969) "A Study on Sampling and Isolation Methods for the Detection of Virus in Sewage," Water Research3 Pergamon Press, 3, pp. 823-832. Lund, E. (1970) Personal communication. The Royal Veterinary and Agricultural University of Copenhagen, Department of Virology, Bulowsve-13, 1870 Copenhagen V Denmark. Mack, W. N., Mailman, W. L., Bloom, H. H., and Krueger, B. J. (1958) "Isolation of Enteric Viruses and Salmonellae from Sewage. I. Comparison of Coliform and Enterococci Incidence to the Isolation of Viruses," Sewage and Industrial Wastes^ 30:8, pp. 957-962. Mack, W. N., Frey, J. R., Riegle, B. J., and Mailman, W. L. (1962) "Enterovirus Removal by Activated Sludge Treatment," Journal Water Pollution Control Federation;^:!!, pp. 1133-1139. McHale, J. S., Hardy, K. J., and Sweet, B. H. (1970) "Concentration of Virus from Water by Forced-Flow Electrophoresis and Electro- Osmosis. Bacteriol. Proceedingst G108, p. 31. ------- -65 Melnick, J. L., Emmons, J., Opton, E. M., and Coffey, J. H. (1954) "Coxsackie Viruses from Sewage: Methodology Including an Evaluation of the Grab Sample and Gauze Pad Collection Procedures," American Journal of Hygiene3 59, pp. 185-195. Metcalf, T. G. (1970) Personal communication. Department of Microbiology, University of New Hampshire, Durham, New Hampshire 03824. Moore, M. L., Ludovici, P. Pa, and Jeter, W. S. (1970) "Quantitativfe Methods for the Concentration of Viruses in Wastewater," Journal Water Pollution Control Fsderation3 Part 2, 42:2, pp. R21-R28. Nupen, E, M. (1970) "Virus Studies on the Windhoek Waste-Water Reclamation Plant (South Africa)," Water Research, Pergamon Press, 4, pp. . Nupen, E. M. (1970) Personal commmication. National Institute for Water Research, P. 0. Box 395, Pretoria, South Africa. philipson, L., Albertsson, P. A., and Frick, G. (1960) "The Purification and Concentration of Viruses by Aqueous Polymer Phase Systems," Virology3 11, pp. 553-571. Poynter, S. F. B. (1970) Personal communication. Metropolitan Water Board. The Laboratories, New River Head, 177 Rosebery Avenue, London, E.C.I,, England. public Health Service Drinking Water Standards, 1962. U, S. Department Health, Education, and Welfare, Washington, D. C, pan, N. U., and Labzoffsky, N. A. (1969) "A Simple Method for the Detection of Low Concentration of Viruses in Large Volumes of Water by the Membrane Filter Technique," Canadian Journal of Microbiology3 15:5, pp. 399-403. Rao, V. C., Sullivan, R., Read, R. B., and Clarke, N. A, (1968) "A Simple Method for Concentrating and Detecting Viruses in Water," Journal American Water Works Association, 60:11, pp. 1288-1294. Stao, V. C. (1970) Personal communication. Central Public Health Engineering Research Institute,'Nagpur 3, India. / Reed, L. J., and Muench, H. (1938) "A Simple Method of Estimating Fifty Percent Endpoints," American Journal of Hygiene, 27, pp. 493-497. ------- -66 Sabin, A. B. (1932) "Experiments on the Purification and Concen- tration of the Virus of Poliomyelitis," Journal Eccperimental Medicine, 56, pp. 307-317. SchSfer, E. (1970) Public Health Aspects of Viruses in Water," N.A. Clarke, ed, Cincinnati, Ohio, 1, p. 14. SchMfer, E., and Borneff, J. (1970) "Evaluation of the FeCl^- Flocculation-Method Combined with the Two Phase-System for Concentrating Viruses," Personal communication. Institute of Hygiene, Virus Laboratory University, 65-Mainz/Germany and Institute of Military Medicine and Hygiene, Virology Department, 54-Koblenz/Germany. Schwerdt, C. E. (1965) "Chemical and Physical Methods," in Viral and Rickettsial Infections of Man, F. L. Horsfell and I. Tamm, ed., Philadelphia: J, B. Lippincott Company, Shuval, H. I., Cymbalista, S,, Fattal, B., and Goldblum, N. (1967) "Concentration of Enteric Viruses in Water by Hydro-Extraction and Two-Phase Separation" in Transmission of Viruses by th-s Water Route, G. Berg, ed. Interscience Publishers, New York. Shuval, H. I., Fattal, B., Cymbalista, S,, and Goldblum, N. (1969) "The Phase-Separation Method for the Concentration and Detection of Viruses in Water," Water Research, Pergatnon Press, 3, pp. 225-240. Stevenson, R. E., Chang, S. L,, Clarke, N. A., and Kabler, P. W. (1956) "Concentration of Dilute Virus Suspensions by Alum Flocculation," Proceedings Society Experimental Biology and Medicine, 92, pp. 764-767. Ver, B. A., Melnick, J. L., and Wallis, C. (1968) "Efficient Filtration and Sizing of Viruses with Membrane Filters," Jourcal of Virology3 2:1, pp. 21-25. "Viruses in Water" (1969) Clarke, N. A. (Chran), Committee on Viruses in Water Report, Jouvaal American Water Works Association} 61:10, pp. 491-494. Wallis, C., and Melnick, J, L. (1967a) "Concentration of Enteroviruses on Membrane Filters," Journal of Virology, 1:3, pp. 472-477. Wallis, C., and Melnick, J. L. (1967b) "Concentration of viru®®sTTfr°m, Sewage by Adsorption on Millipore Membranes," Bulletin Wor a :zealtr. Organization, 36, pp. 219-225. ------- -67 Wallis, C., Melnick, J. L. (1967c) "Concentration of Viruses on Aluminum and Calcium Salts," American Journal of Epidemiologyi 85:3, pp. 459-468. Wallis, C., Grinstein, S., Melnick, J. L., and Fields, J. E. (1969) "Concentration of Viruses from Sewage and Excreta on Insoluble Polyelectrolytes," Applied Microbiologyj 18:6, pp. 1007-1014. Wallis, C., Melnick, J. L., and Fields, J. E. ( ) "Detection of Viruses in Large Volumes of Natural Waters by Concentration on Insoluble Polyelectrolytes, Water Eeseca,dhi Pergamon Press (accepted for publication September 17, 1970), Witt, G. (1965) "Nachweis von Menschenpathogenen Viren im Trinkwasser nach Anreicherung mittels Hochdruckfiltration durch ein lOsliches TJltrafilter," Abstract Zentralblatt fUr Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene, 198, pp, 161-162, ------- |