Polyelectrolytes. and penicillin-streptomycin mixture, 100 units or 100 iag per ml). Viruses were harvested from serum-free

Transcription

1 APPuLE MICROBIOLOGY, Apr. 1971, p American Society for Microbiology Vol. 21, No. 4 Printed in U.S.A. Concentration and Purification of Viruses by Adsorption to and Elution from Insoluble Polyelectrolytes CRAIG WALLIS, JOSEPH L. MELNICK, AND JOSEPH E. FIELDS Department of Virology and Epidemiology, Baylor College of Medicine, Houston, Texas 7725, and Monsanto Co., St. Louis, Missouri Received for publication 31 December 197 Acid-resistant, nonenveloped viruses belonging to the enterovirus, reovirus, and adenovirus groups ere readily concentrated on PE6, an insoluble cross-linked polyelectrolyte based on isobutylene maleic anhydride. Hydrolysis of PE6 by NaOH increased its capacity to adsorb viruses. Hydrogen ion levels played an important role in virus concentration; optimal ph levels for maximal virus adsorption ere beteen ph 3. and 4.5. Undiluted virus as easily concentrated from large volumes on PE6, and the adsorbed virus as readily eluted at ph 8 to 9. Divinylbenzene cross-linked styrene/maleic anhydride copolymers ere found to adsorb highly purified tobacco mosaic virus (TMV) and poliovirus (3). Purified TMV as adsorbed to polyelectrolytes in salt solution, hereas purified poliovirus as adsorbed to these polymers in distilled ater. The effect of organic material on the adsorption of viruses to the polymers as not investigated in the preliminary study by Johnson, Fields, and Darlington (3). Viruses as found in nature or as gron in the laboratory are present in fluids along ith salts and organic material. Therefore, conditions for the concentration of viruses from natural or laboratory-gron fluids require more definitive data as to the role of organic and inorganic factors in the adsorption of viruses to polyelectrolytes. MATERIALS AND METHODS Monkey kidney (MK) cells. Kidneys obtained from immature green (vervet) monkeys ere trypsinized and gron in Melnick's medium A containing 2% calf serum. Viruses. A plaque-purified line of virulent type 1 poliovirus (Mahoney) ith a titer of 1' plaqueforming units (PFU)/ml as used for most of these studies. Other agents used ere representative viruses from a number of different taxonomic groups. These consisted of plaque-purified lines of type 1 attenuated poliovirus (LSc vaccine strain); echovirus types 1 (Farouk strain), 7 (Wallace strain), and 12 (Travis strain); coxsackievirus types A9 (Grigg strain) and B3 (Nancy strain); adenovirus (SV15 st:ain); and reovirus type 1 (716 strain). Virus stocks ere prepared by a single-cycle passage in MK cultures maintained ith Melnick's medium B (.5% lactalbumin 73 hydrolysate, Earle's salt solution,.22% NaHCO3, and penicillin-streptomycin mixture, 1 units or 1 iag per ml). Viruses ere harvested from serum-free cultures and from cultures maintained on media hich do not contain detergent. Serum or detergent (such as Teen 8 used in medium 199) elute virus adsorbed to PE6. Virus assays. Quantification of virus as performed by the PFU method. Overlay medium consisted of Earle's salt solution,.4% NaHCO3, 1: 6, neutral red, amino acids as used in Eagle's minimal essential medium, and 1.5% agar (Difco). To enhance plaque formation, 25 mm MgCl2 as included in overlays for enteroviruses (9), 4,g of protamine sulfate per ml as used in overlays for adenovirus (9), and 1:6 pancreatin (Oxoid) in overlays for reovirus (11). Insoluble polyelectrolyte 6 (PE6). PE6, available from the Monsanto Co., St. Louis, Mo., as provided in the form of a 1-mesh poder. It is an insoluble cross-linked copolymer of isobutylene maleic anhydride. Preparation of PE6. The polyelectrolyte (PE) as prepared for use by making a 1% suspension in distilled ater. The suspension in a centrifuge bottle as shaken on a rotary shaker for.5 hr; the vessel as then centrifuged at 2, rev/min for 5 min, and the supernatant fluid as discarded along ith a small amount of nonsedimentable, or nonettable, product. The packed PE as then ashed tice ith distilled ater. After the last ashing, the pelleted PE as resuspended in.1 volume of distilled ater to make a 1% suspension. The suspension in portions of 1 ml as then distributed into tubes; the tubes ere centrifuged as described, and the supernatant fluids ere discarded. The packed PE, 1 mg per tube, as treated by addition of a given volume of virus to the tube, or the packed PE as transferred to virus fluids by interchange of virus fluid into the packed PE tube Donloaded from on November 12, 218 by guest

2 74 WALLIS, MELNICK, AND FIELDS APPL. MICROBIOL. and back to the vessel used for stirring or shaking the virus-pe mixture. Tubes or beakers containing packed PE ere treated ith virus fluids on a rotary shaker or on magnetic stirrers to afford a good collision efficiency beteen the virus and the PE. After 1 hr of rotation or stirring at 25 C, the virus-pe mixtures ere centrifuged at 2, rev/min for 5 min, and the supernatant fluids ere obtained and assayed for unadsorbed virus. Borate buffer. A.5 M solution of sodium borate as made in distilled ater and adjusted to ph 9.. RESULTS Adsorption of poliovirus to hydrolyzed PE6. Poliovirus harvests diluted 1,-fold in distilled ater, saline, or phosphate buffer (ph 6 or ph 7) avidly adsorbed to PE6 (hich had been treated by ashing ith ater as described in Materials and Methods). On the other hand, poliovirus did not adsorb to PE6 in buffers above ph 8 or in any diluent containing serum or tissue culture extracts, regardless of ph. Thus, poliovirus ill adsorb to PE in diluents free from proteins at neutral or acid ph levels. Experiments ith PE6 and undiluted poliovirus at different ph levels (2.5 to 8) failed to give any significant adsorption of this virus to the PE, apparently because of the nonviral proteins present in the harvest fluids. During the course of these experiments, it as Uz I- >1 z \ Z S : N8OH- WASHE 2 3 L Donloaded from on November 12, 218 by guest noted that PE6 suspended in ater, stored at 25 C, and used from day to day began to improve in its ability to adsorb undiluted virus at lo ph ranges. Such an increase in the number of reactive sites for virus adsorption could result from slo hydrolysis of the PE during storage. Therefore, freshly prepared PE6 as treated ith HCI or NaOH to expedite hydrolysis. The adsorption of poliovirus to hydrolyzed PE6 is shon in Fig. 1, hich is typical of several experiments that ere carried out. Poliovirus did not effectively adsorb to ater-ashed PE6 at ph 2.5 to 8.. PE6 treated ith HCI gave results almost identical to those obtained ith ater-ashed PE, and these data ere omitted from the figure for the sake of clarity. Hoever, NaOH-ashed PE adsorbed virus from suspension ith increased avidity. Repeated experiments indicated that the optimal ph range for virus adsorption as ph 2.5 to 4.5 for NaOH-ashed PE6; consequently, ph 3.5 as used for subsequent experiments. A number of parameters ere examined to determine the optimal degree of PE6 hydrolysis for virus adsorption. As shon in Fig. 2, treatment of PE6 ith NaOH as most effective beteen.3 and.3 N. When the NaOH concentration as increased to 3 N, virus as no longer adsorbed to the hydrolyzed PE. The.5-hr treatrnent time used in the above exph OF POLIOVIRUS- POLYELECTROLYTE MIXTURES FIG. 1. Adsorption ofpoliovirus to hydrolyzed PE6. One gram of PE6 as ashed ith 5 ml of.1iv NaOH for.5 hr on a rotary shaker. The PE as then packed at 2, rev/min for 5 min and repeatedly ashed (five times) ith 2 ml of distilled ater to remove residual NaOH. The suspended PE in distilled ater as then distributed into tubes giving a concentration of 1 mg of PE per tube. The tubes ere centrifuged, the ater as discarded, and the packed PE as treated ith 5 ml of undiluted virus for I hr on a shaking machine at the ph indicated (HCI or NaOH as used to adjust ph levels). After I hr of mixing, the tubes ere centrifuged, and the supernatants ere obtained and assayedfor unadsorbed virus. Control virus (PE-free) at the ph levels indicated shoed no decrease in infectivity. A duplicate test as described above as performed ith PE ashed only ith distilled ater (ater-ashed PE).

3 VOL. 21, 1971 CONCENTRATION AND PURIFICATION OF VIRUSES 75 U \ L \ o\ J3 o \ /~~~~~ v 4.4,. ODOIlN O.OIN O.IN l.on NaOH CONCENTRATION USED FOR PE6O TREATMENT FIG. 2. Effiects of NaCH concentration on hydrolysis of PE6O. Samples (1 mg) of PE6O ere treated ith S ml of NaOH at the concentration indicated for.5 hr at 25 C. The NaOH-ashed PE preparations ere then repeatedly ashed ith distilled ater to remove residual base, andl the packed PE preparations (1 mg per tube) ere then treated ith S ml of undiluted poliovirus at ph 3.5 for 1 hr on a rotary shaker. The tubes ere then centrifuged, and the supernatant fluids ere assayed for unadsorbed virus. Control virus held at ph 3.5 for 1 hr at 25 C on the rotary shaker did not lose titer. periments had been arbitrarily chosen. To determine the effects on PE6 of different times of treatment ith NaOH, 1-mg samples of PE6 ere treated ith 5 ml of.1 N NaOH at 25 C for the times indicated in Fig. 3. The results of this experiment indicate that treatment ith NaOH for more than.5 hr did not greatly increase virus attachment to the PE. Hoever, prolonged treatment (5 hr or longer) produced excess hydrolysis, and less poliovirus as adsorbed to the hydrolyzed products. Repeated experiments confirmed the above results and indicated that.5- to 3.5-hr NaOH treatment of PE6O at 25 C gave essentially the same results. Since hydrolysis proceeds more rapidly at elevated temperatures, the effects of increased temperatures ere determined. PE6 (1-mg samples) as treated ith.1 N NaOH for.5 hr at 4, 25, 37, 5, 6, 7, and 8 C. The NaOHtreated PE preparations ere then ashed five times ith large volumes of distilled ater to remove residual NaOH. A 5-ml amount of undiluted poliovirus as added to each 1 mg of packed PE, and the ph as adjusted to 3.5 ith HC1. The samples ere rotated mechanically for 1 hr. The tubes ere centrifuged at 2, rev/min for 5 min, and the supernatant fluids ere assayed for infectivity. As shon in Fig. 4, treatment of the PE at temperatures over 6 C caused excess hydrolysis, and less virus as removed. Lo temperatures (4 and 25 C) did not produce sufficient hydrolysis; the optimal temperature for NaOH treatment of the polymer as 37 to 55 C. Elution of poliovirus from PE6. Since poliovirus adsorption to PE6 as negligible in isotonic salt solutions at high ph levels, or in the presence of serum, such diluents ere used to elute adsorbed virus. The test as performed by adsorbing poliovirus to NaOH-ashed PE6, and then the PE-v'rus complex as distributed into tubes so that each tube contained 1 mg of PE and 5 X 18 PFU of poliovirus. The packed PEvirus complex as treated ith different solvents, and the eluents ere assayed. The experimental procedures and results of these tests are described in Table 1. Eluents used at lo ph levels (7.5 and belo) failed to elute the virus. Hoever, virus could be efficiently recovered from the PE ith phosphate buffer at ph 8 or ith borate buffer at ph 9. Thus, virus can be efficiently recovered ith eluents that by themselves are nonantigenic. The elution efficiency depended on the ph achieved after the eluent as added to the packed PE. Borate at ph 9 added to a small amount of packed PE as affected by the acidic polymer, and ph levels of the PE-eluent mixture averaged 7.5 to 8.5. When 3 ml of borate buffei as added to 1 g of packed acidic PE hich had O 4 s R/ TIME IN HOURS FOR PRETREATMENT OF PE6 WITH NaOH AT 25-C FIG. 3. Effect of time of NaOH treatment on PE6. Same procedure as described in Figure 2, except that.1 N NaOH as used to treat PE6 for different periods of time at 25 C. Donloaded from on November 12, 218 by guest

4 76 WALLIS, MELNICK, AND FIELDS APPL. MICROBIOL. cn z -, 2 _ U) M :i4 -c o-c 2-C 4'C 6IC OC TEMPERATURES USED FOR PESO TREATMENT FIG. 4. Effect of temperature on the hydrolysis of PE6. Same methods as described in Fig. 2, except that PE6 as treated ith.1 N NaOH for.5 hr at the temperatures indicated ithout shaking. NaOH as added to the PE, hich had been suspended by shaking a fe times, and then the PE-NaOH mixture as placed at the temperatures indicated. A control of PE6-NaOH mixture hich as shaken for.5 hr on the rotary unit as compared ith a duplicate sample hich as stored at 25 C ithout shaking. Both samples yielded similar results, and therefore only one sample at 25 C is plotted. been used to adsorb virus at ph 4, the eluate as brought to acidic ranges, and elution as poor. Therefore, after addition of eluents to the PE, the mixture as monitored and readjusted to ph 9 to obtain efficient elution. Effect of time and PE concentration on poliovirus adsorption. Under the optimal conditions for hydrolysis of the PE as described above, an experiment as conducted to determine the capacity of 1 mg of PE to adsorb excess virus. In duplicate experiments, the reaction of 4 mg of PE6 ith 2 ml of undiluted virus as compared ith the reaction of the virus ith 25% of the amount of PE previously used (1 mg/2 ml of virus). The time as varied, as shon in Fig. 5. Although the 4 mg of PE6 used to adsorb the virus contained in the 2 ml of undiluted harvest removed virus from the supernatant more effectively, the 1 mg concentration adsorbed 99% of the virus ithin the first hour. When 1 ml of undiluted virus as treated ith 1 mg of PE6 for 2 to 4 hr, again 99% of the virus as adsorbed. This avoided the use of excess PE hich ould require undesirably large volumes of eluate. Concentration of large volumes of poliovirus on PE6. Under the optimal conditions for adsorbing poliovirus to PE6 as described above, 1 liter of poliovirus (undiluted harvest containing 11 PFU per ml) as concentrated, as shon in Fig. 6. PE6 (1 g) as added to 1 liter of virus at 25 C, the ph as adjusted to 3.5, and the mixture as stirred gently for 4 hr. The titer of unadsorbed virus recovered as 5.7 log1o, indicating adsorption of greater than 99% of the total virus population. Virus eluted from the PE reflected a TABLE 1. Elution of poliovirus from PE6 Logio Poliovirus treatmenta titer or eluted ml) Control, untreated virus Virus-PE supernatant >99.99% adsorbed Eluents Eluate titer Distilled ater, ph <2. % eluted Saline, ph 6.8.<2. % eluted Phosphate buffer,b ph % eluted Borate buffer % eluted (.5 M), ph 9.. Fetal serum (1%) in saline, ph % eluted Fetal serum (1%) in ph 8 phosphate % eluted a Undiluted poliovirus (3 ml) as added to 6 mg of PE6 hich had been ashed ith NaOH as described above. The suspension as adjusted to ph 3.5 ith HCl, and then as placed on a magnetic stirrer for 1 hr at 25 C. The suspension as centrifuged at 2, rev/min for 5 min, and the supernatant fluid as collected for assay. The packed PE-virus complex as resuspended in 3 ml of distilled ater, and 5 ml of the ell-mixed suspension as distributed into each of six tubes, giving a final concentration per tube of 1 mg of PE and 5 X 18 PFU of poliovirus bound to PE. The tubes ere centrifuged, and the supernatant fluids ere discarded. The packed PE-virus complex as treated ith 5 ml of the eluents indicated by vigorously shaking the PE-eluent mixture for a fe seconds. The ph of the PE eluent as checked on a ph meter, and adjustments ere made ith NaOH to attain the original ph of eluent. The mixture as then centrifuged as described above, and the supernatant fluids ere assayed for eluted virus. bsorensen's phosphate, NaH2PO4,.5 M. Donloaded from on November 12, 218 by guest

5 VOL. 21, 1971 CONCENTRATION AND PURIFICATION OF VIRUSES 77 z oi a. =2 \ \ > - 3. LL 4_/ F ~~~~\ 1 MG PE6 4 MG PE6 Z W 5 C'\ 6 o\ 7_ 8 It4 1/2 1 2 TIME IN HOURS OF STIRRING VIRUS-PE MIXTURE AT FIG. 5. Effiect of time and of FE6O concentratrion. Filtrate FE6O hydrolyzed ith.1 N NaOH at 37 C fori2hrl as ashed ith distilled ater and used to concent'rate poliovirus as follos: 2 ml of virus as mixed ith 1 mg offe6 andl a second sample of 2 ml of virus as mixed ith 4 mg of FE6. The ph as adju Trapped PE recovered sted and suspended in 3 ml to 3.5 ith HCl, and the samples ere stirred a)n a of eluent magnetic unit for the time indicated. Samples ere obtained and centrifuged to sediment the FE. supernatants ere assayed for unadsorbed virus. Eluate 6% recovery and a 2-fold concentration ml Ċounterimmunoelectrophoresis gave precip lines at a dilution of 1 :4 ith the original v harvest against antiserum made in rabbits aga normal tissue culture extracts (MK). The 2 concentrate (eluate) failed to manifest a precip line even hen tested ithout dilution against same antiserum. Thus, although the virus concentrated 2 times, the nonviral protz ere not concentrated, as they ere not detecti in the eluate under the conditions of the test. The adsorption of virus to the PE as enhan by the filtration procedure used (see Fig. Virus that had not collided ith PE and becc adsorbed during stirring as passed through a pad, thus increasing the efficiency of virus adse tion. 25"C Concentration of other acid-resistant viruses on PE6. A number of other acid-resistant viruses ere treated ith PE6 as described above. The experimental procedures and results are shon in Table 2. All viruses tested (coxsackievirus, echovirus, reovirus, and adenovirus) ere adsorbed to the PE, and efficient elution of the viruses as accomplished in.1 volume. Thus, all viruses examined could be concentrated on and eluted from PE6 by use of a simple procedure. Since acid conditions are required for maximal virus adsorption, conditions must be orked out 1 liter of undiluted poliovirus +1 g of NaOH-hydrolyzed PE6, stirred 4 hr at ph 3.5, 25 C Filter through coarse pad to trap the PE ASSAYS Logi PFU/ml ) 1.3 FIG. 6. Concentration of poliovirus from large volumes on PE6. One liter of undiluted poliovirus as treated ith I g of NaOH-treated PE6 at ph 3.5 for 4 hr at 25 C on a magnetic stirrer. The virus-pe mixture (hile still stirring) as then filtered (at 25 C) through an AP2 coarse fiberglass pad (Millipore Corp.) to trap the virus-pe complex on the filter pad. This as performed by leading a rubber tubing from an aspirator bottle containing the PE-virus suspension to the inlet ofa 47-mm Millipore filter holder containing the AP2 filter pad. The outlet of the filter holder as connected to a vessel to hich vacuum as applied. After the entire suspension passed the pad, the filter holder as opened, and the virus-pe as scrapedfrom the pad ith a spatula. The recovered PE as suspended in 3 ml of borate-naoh buffer (ph 9,.5 M) to elute the virus. After vigorous mixing of the PE-eluent mixture, the supernatant as assayed for virus. The total time requiredfor this procedure as less than 4.5 hr. Donloaded from on November 12, 218 by guest

6 78 WALLIS, MELNICK, AND FIELDS APPL. MICROBIOL. TABLE 2. Concentration of other acid-resistant viruses on PE6 Logio virus titer (PFU/ml) of No. of Virusesa Virus-polyelectrolyte mixture times concen- Control trated Supernatant Eluate Type 1 poliovirus, LSc Echoviruses Type < Type Type < Coxsackieviruses A9 7.7 < B < Reovirus, type Adenovirus, SV a Undiluted virus (5 ml) as treated ith 1 mg of PE6 (preashed ith NaOH as described) for 1 hr at 25 C at ph 3.5 on a magnetic stirring apparatus. The suspensions ere centrifuged at 2, rev/min for 5 min, and supernatant fluids ere assayed for unadsorbed virus. The packed PE as treated ith.5 ml of borate buffer (ph 9.) by vigorously mixing the PE-eluent mixture for a fe minutes, and the supernatant fluids ere assayed for eluted virus. for acid-sensitive viruses (e.g., herpesvirus, myxovirus, vaccinia virus, and rhinovirus). DISCUSSION The concentration of acid-resistant viruses (enteroviruses, reoviruses, and adenoviruses) on exchange resins or adsorbents has not been practical as a routine laboratory procedure. In many cases, the methods employed are laborious and complicated. For example, the concentration of poliovirus on exchange resins (Doex-1, anion) required that the virus harvest be exhaustively dialyzed (for 3 days) to remove interfering components that ould exchange on the resin and prevent virus exchange (5), and then only 2.25 ml of virus could be passed through the resin per hour to obtain efficient concentration. The adsorption and elution of 1 enteroviruses to bentonite as dependent on a number of requirements (4): three strains ere adsorbed at acid ph levels, but the degree of adsorption varied. Elution of the viruses required different solutions, and thus no single procedure as established for concentration and elution of the 1 viruses tested. Similarly, adenovirus concentration on diethylaminoethyl-cellulose columns required dialysis of the virus stocks, and then elution of exchanged virus at specific molarities of salts (2). Different serotypes required different molarities for elution off the column. In the current study, e have shon that acidresistant viruses preferentially adsorb to an insoluble PE in the presence of tissue culture proteins, salts, amino acids, and vitamins, ultimately yielding a virus concentrate in the form of an eluate lo in nonviral protein. The method is simple; the requirements for adsorption to and elution from the PE of all viruses tested are essentially identical. Although each of the tables and figures describes a single experiment, the data are representative of many experiments carried out ith each virus, hich yielded essentially the same results. NaOH-treatment of PE enhanced adsorption of certain viruses, but such hydrolysis did not deleteriously affect the PE and did not prevent adsorption of those viruses hich do not require NaOH treatment of the PE. Thus, hydrolysis of PE6 could be routinely performed so that all acid-resistant viruses could be concentrated by a single procedure. These methods have been adapted to routine use, and Table 3 indicates the parameters for concentration on PE6 of acid-resistant viruses present in different fluids. Table 3 also indicates the conditions for concentration of influenza viruses. TABLE 3. Optimal conditions for adsorption ofacidresistant viruses and influenza viruses to PE 6 Optimal ph Virus fluids PE treatment of PE-virus Reference mixture Acid-resistant viruses In tissue culture harvests. In seage. In tap aters.. In feces and urine. Influenza PR 8 strain in allantoic fluid. Hong Kong strain in allantoic fluid NaOH Water only Water only Water only Water onlyl b NaOH bI Current study _C -c a In the absence of excess organics and in hypotonic fluids, PE6 adsorbs viruses at a ide ph range (1). bviruses stabilized against deleterious effects of excess hydrogen ions ith ammonium sulfate (A. Homma et al., in preparation). * A. Homma et al, in preparation. Donloaded from on November 12, 218 by guest

7 VOL. 21, 1971 CONCENTRATION AND PURIFICATION OF VIRUSES 79 The concentration of viruses on collodion membranes (8) is more laborious and requires more equipment than the procedures described in the present paper. Adsorption of viruses to aluminum salts (7), also used for virus concentration, presents a problem in that only limited quantities of fluids can be used before the alumina gel clogs the filtration system; further, the tissue culture harvests must be diluted 1-fold before use to reduce the protein concentration of the virus suspension because A12(OH)3 binds proteins and depletes the available sites for virus adsorption. One application of this study is the purification of viruses by adsorption to and elution from polyelectrolytes in the preparation of vaccines of good antigenicity ith lo reactivity as the nonviral proteins are removed. Another application is in the detection of viruses contained in natural fluids here they are often present in subinfectious quantities. Concentration of virus on PE6 from seage, urine, feces, and tap aters has already been reported (1, 6, 1). ACKNOWLEDGMENT This investigation as supported by a grant from the Monsanto Co., St. Louis, Mo. LITERATURE CIED 1. Grinstein, S., C. Walls, and J. L. Melnick Virus isolations from seage and from a stream receiving effluents of seage treatment plants. Bull. W.H.O. 42: Haruna, I., H. Yaoi, R. Kono, and I. Watanabe Separation of adenovirus by chromatography on DEAE-cellulose. Virology 13: Johnson, J. H., J. E. Fields, and W. A. Darlington Removing viruses from ater by polyelectrolytes. Nature (London) 213: Shirobokov, V. P Differentiation of coxsackieviruses based on the character of adsorption onto bentonite. Acta Virol. 12: Taylor, J., and A. F. Graham Purification of poliovirus labeled ith radiophosphorus. Virology 6: Waffis, C., S. Grinstein, J. L. Melnick, and J. E. Fields Concentration of viruses from seage and excreta on insoluble polyelectrolytes. Appl. Microbiol. 18: Wallis, C., and J. L. Melnick Concentration of viruses on aluminum and calcium salts. J. Epidemiol. 85: Wallis, C., and J. L. Melnick Concentration of enteroviruses on membrane filters. J. Virol. 1: Wallis, C., and J. L. Melnick Mechanism of enhancement of virus plaques by cationic polymers. J. Virol Wallis, C., J. L. Melnick, and J. E. Fields Detection of viruses in large volumes of natural aters by concentration on insoluble polyelectrolytes. Water Res., in press. 11. Wallis, C., J. L. Mehnick, and F. Rapp. Effects of pancreatin on the groth of reovirus. J. Bacteriol. 92: Donloaded from on November 12, 218 by guest