Concentration and Purification of Influenza Virus on Insoluble Polyelectrolytes

APPEuw MicRoBIoLoGY, Apr. 1972, p. 740-744 Copyright 0 1972 American Society for Microbiology Vol. 23, No. 4 Printed in U.S.A. Concentration and Purification of Influenza Virus on Insoluble Polyelectrolytes CRAIG WALLIS, AKIRA HOMMA, AND JOSEPH L. MELNICK Department of Virology and Epidemiology, Baylor College of Medicine, Houston, Texas 77025 Received for publication 11 January 1972 A method for rapid concentration and purification of influenza virus by adsorption on and elution from an insoluble polyelectrolyte is described. To accomplish this task, influenza virus had to be rendered stable at ph 4 to 5, since viruses adsorb to the polyelectrolyte more efficiently at this ph range. A precipitate which forms in influenza harvests under acid conditions in the cold can be removed by ammonium sulfate at a concentration which traps the precipitate but not the virus. Thus, ammonium sulfate-treated influenza virus in allantoic fluid could be readily concentrated on the polyelectrolyte. Elution yielded a virus concentrate essentially free of nonviral proteins. In a recent series of reports (3, 13, 15), we have shown that a newly available polyelectrolyte is capable of adsorbing enteroviruses from whole virus harvests without adsorbing significant amounts of nonviral proteins. The current report is concerned with the application of the polyelectrolyte method to the concentration and purification of influenza viruses, which could be used for vaccine production. The virus concentration and purification was measured by hemagglutination activity. MATERIALS AND METHODS Influenza virus. The PR-8 strain was used as a model; it was obtained from the American Type Culture Collection, Washington, D.C. This strain has been through 150 serial passages in 10- to 11-day-old embryonated eggs. The stock used in this study was made by allantoic inoculation with 10' infectious doses. After 48 hr of incubation at 36 C, surviving embryos were chilled and allantoic fluids were harvested. Pooled allantoic fluids were frozen at -70 C until use, representing a storage period of 1 month. They were then thawed at 36 C, vigorously shaken to disperse the precipitate, and clarified before use by centrifugation at 2,000 rev/min for 15 min. The supernatant fluids were used as the virus stocks. HA tests. Serially diluted virus (0.5-ml volume) was mixed with 0.5 ml of thrice-washed 0.5% guinea pig erythrocytes in saline, and hemagglutination (HA) patterns were scored after 2 hr at 25 C. PE 60. The insoluble polyelectrolyte (PE) was provided by and is available from the Monsanto Company, St. Louis, Mo.; it is a cross-linked copolymer of isobutylene maleic anhydride. It was made available as a 100-mesh powder and was prepared for use as follows. A 1% suspension was made in distilled water and shaken on a rotary unit for 30 min. After centrifugation at 2,000 rev/min for 5 min, the supernatant fluids containing a small amount of nonwettable polymer were discarded. The packed PE was then washed twice in distilled water, and after the last washing, the PE was resuspended in distilled water to a final volume of 100 ml. Each 10 ml of this suspension contained 100 mg. The required amount of PE was distributed to tubes and centrifuged; then virus was added with mixing to the packed PE. We have shown (3, 13, 15) that enhanced adsorption of enteroviruses was achieved with PE 60 if it was first treated with NaOH. However, experiments with influenza virus revealed that conditioning the PE with NaOH had no advantages. Thus, only water-treated PE was used in this study. RESULTS Partial purification of influenza virus with ammonium sulfate. In previous studies with enterovirus and reovirus concentration on PE 60 (3, 13, 15), it was demonstrated that virus adsorption occurred only at acid ph levels when virus was suspended in organic medium (i.e., tissue culture harvests, sewage effluents, human excreta, etc.). Experiments with influenza virus as harvested in undiluted allantoic fluids revealed that this virus also efficiently adsorbed to PE 60, only at ph levels below neutral. Because of the acid sensitivity of influenza virus, the experiments were performed at 4 C. At this temperature we encountered a precipitate in the allantoic fluid that ripens upon standing in the cold at ph levels of 6.5 or lower. Since influenza virus is known to be sensitive to acid levels below ph 6.0, the adsorption was conducted at ph 6.0. At this 140

VOL. 23, 1972 CONCENTRATION OF INFLUENZA VIRUS 741 ph, only 50% of the virus was adsorbed. Further difficulties arose upon centrifugation (2,000 rev/min for 5 min) of the PE to concentrate the virus, for the cold-acid precipitate was also sedimented. Elution of virus from the PE under these conditions results in an eluate contaminated with this allantoic precipitate. When the precipitate at ph 6.0 in the absence of PE was ripened at 4 C and the material was centrifuged, tests of the supernatant fluids revealed that a large proportion of influenza virus, as measured by HA activity, had also been removed with the sediment. This virus apparently complexed with the precipitate during its formation. Experiments were therefore designed to partially purify influenza virus by precipitating out the component which was responsible for the acid-cold precipitate, without sedimenting the virus. Undiluted influenza virus stock in the form of allantoic fluid harvests was treated with ammonium sulfate at different concentrations for 15 min. A precipitate formed which was removed by centrifugation, and the supernatant fluids were assayed for HA activity. This test was performed to determine the lowest concentration of ammonium sulfate which removed the cold-acid precipitate without affecting the virus HA titer. To determine whether the factor in the egg fluids which precipitated at acid levels in the cold had been removed, portions of the supernatant fluids described above were adjusted to ph 5.5 (where the precipitate rapidly forms) and held in an ice bath. The degree of turbidity was recorded. The experimental procedures and results of this test are shown in Table 1. At final concentrations of 40% saturated ammonium sulfate or higher, virus was removed from solution. However, at 30% saturated ammonium sulfate or lower, no detectable HA activity was removed by the sulfate. The control virus sample (treated with water in lieu of sulfate) held for 3 hr at ph 5.5 manifested a massive precipitate. The precipitate was not evident in samples treated with ammonium sulfate at final concentrations of 10% or higher saturation. Stabilization of influenza virus by ammonium sulfate in acid medium. Since the acidcold precipitate could be removed by ammonium sulfate precipitation, the role of ammonium sulfate on the stability of influenza virus at acid ph levels was next investigated, since viruses adsorb more efficiently to PE 60 under acid conditions. Undiluted PR-8 virus (as present in whole allantoic fluid) was treated with saline or a final concentration of 10% saturated ammonium sulfate, and the ph was adjusted from 4.0 to 8.0. After 3 hr at 4 C, the degree of precipitation was recorded and HA activity was assayed. The results are shown in Table 2. Precipitates were formed in ammonium sulfate-free samples at ph 6.0 and in- TABLE 1. Partial purification of influenza virus with ammonium sulfate Final concn of saturated HA titer of Precipitates ammonium sulfatea supernatant formed at (%) fluids ph 5.5b None, saline control 640 Gross 50 80 None 40 320 None 30 640 None 20 640 None 10 640 None 5 640 Slight 2.5 640 Gross a Undiluted virus stock in allantoic fluid was mixed with an equal volume of saline (0.15 M NaCl) or with saturated ammonium sulfate (at 25 C) to give the final concentrations indicated. Samples were shaken on a rotary unit for 15 min at 25 C, and tubes were then centrifuged at 3,000 rev/min for 5 min to sediment precipitates. The supernatant fluids were then assayed for hemagglutination (HA) activity. 'Samples of the supernatant fluids prepared as described in footnote a were adjusted to ph 5.5 and held at 4 C for 3 hr to determine the presence of a precipitate which ripens in the cold at acid ph levels. TABLE 2. Stabilization of influenza virus by ammonium sulfate in acid medium Virus-saline Virus-ammonium ph of mixture sulfate mixture suspension HA Precipi- HA Precipititer tate titer tate 8.0 640 None 640 None 7.0 640 None 640 None 6.0 640 Slight 640 None 5.0 40 Gross 640 None 4.5 < 20 Gross 320 None 4.0 < 20 Massive 160 None a Whole, infected allantoic fluids were treated with saline or ammonium sulfate (nine parts virus plus one part saline or one part saturated solution of ammonium sulfate). Samples were shaken for 15 min on a rotary mixer and centrifuged, and supernatant fluids were obtained and adjusted to the ph levels indicated. After 3 hr of storage at 4 C at the ph range indicated, the degree of precipitation in samples was recorded, and samples were then assayed for hemagglutination (HA) activity (precipitates were removed by centrifugation prior to assay).

742 WALLIS. HOMMA, AND MELNICK APPL. MICROBIOL. creased in amount with lowering of ph. Virus survival in acid medium was inversely related to formation of precipitates. As precipitates formed, HA activity could no longer be detected in the supernatant fluids. These experiments raised the question of whether influenza virus is truly acid-sensitive. The formation of precipitates may bind virus, which would be removed with the precipitate. On the other hand, virus suspended in a final concentration of 10% saturated ammonium sulfate was not precipitated at ph levels as low as 4.0. HA activity was completely stabilized even when the ph decreased to 5.0, and there was significant stability even at ph 4.0. Whether ammonium sulfate directly stabilizes the virus in acid medium or whether the salt precipitates a stabilizing factor is yet to be resolved. Regardless, influenza virus can now be handled at relatively low ph levels in ammonium sulfate salts without deleterious effect. Effects of ph on adsorption of ammonium sulfate-stabilized influenza virus to PE 60. A stock of undiluted PR-8 allantoic fluids was treated with ammonium sulfate to yield a 10% final saturated solution; the precipitate was removed by centrifugation and discarded. The supernatant fluid was adjusted to different ph levels, and PE 60 was added. The virus-pe mixtures were then centrifuged, and the supernatant fluids were assayed for unadsorbed virus. The experimental procedures and results of this test are shown in Table 3. At ph 7.0, there was no significant removal of virus. There was marked adsorption of virus to the PE as the ph reached 5.0. Since the control virus at this ph in the absence of the PE proved to be stable, subsequent adsorption experiments were carried out at ph 5.0. Effects of PE concentration and time on adsorption of influenza virus to PE 60. Experiments described above were arbitrarily performed using 100 mg of PE for each 20 ml of undiluted virus with a stirring time of 1 hr. To determine the optimal parameters for virus adsorption to PE 60, experiments were carried out as shown in Tables 4 and 5. As little as 25 mg of PE 60 was capable of removing 75% of the HA activity in undiluted virus stocks in 1 hr. With 100 mg of PE 60, only 5 min was required to remove 75% of the HA activity. Elution of influenza virus from PE 60. In previous reports we showed that enteroviruse, can be eluted from PE 60 at alkaline ph (3, 13, 15). As shown in Table 3, influenza virus was effectively adsorbed to PE 60 as the ph of the suspension was decreased from neutral to acidic. It seemed, therefore, that influenza TABLE 3. ph of samplesa Effects of ph on adsorption of stabilized virus onto PE 60 HA titers of supernatant fluids from PE-free control PE 60 7.0 1,280 640 6.0 640 320 5.5 1,280 80 5.0 640 <20 4.5 640 <20 4.0 320 <20 a A 180-ml amount of undiluted allantoic fluid containing PR-8 virus was treated with 20 ml of saturated solution of ammonium sulfate. The mixture was shaken at 25 C for 15 min, and then the gross precipitate was removed by centrifugation at 2,000 rev/min for 15 min. The supernatant fluids were obtained, and 20-ml samples were adjusted to ph levels indicated in the table. A 100-mg amount of PE 60 was added, and the samples were stirred for 1 hr at 4 C. Duplicate samples without PE were held at 4 C as a control for hemagglutination (HA) activity at the ph levels tested. After 1 hr of stirring, samples were centrifuged at 2,000 rev/min for 5 min to sediment the PE, and supernatant fluids were assayed for unadsorbed HA activity. TABLE 4. Effects of PE concentration and time on adsorption of influenza virus to PE 60 Concn Time Per cent he- Length of Per cent he- Amt of magglutinin treatment' magglutinen PE 60a unadsorbed of 100 mg unadsorbed (mg) to PE 60 ofpe 60 to PE 60 after 1 hr (min) 100 1 80 1 50 10 40 2 25 25 20 5 12.5 40 10 10 6.25 60 5 25 a Influenza virus partially purified by addition of 10% saturated ammonium sulfate and centrifugation was adjusted to ph 5.0 with HCl, and 20-ml samples were stirred for 1 hr with different amounts of PE. Samples were then centrifuged, and supernatant fluids were assayed for unadsorbed hemagglutination (HA) activity. 'Twenty-milliliter volumes of partially purified virus at ph 5.0 were treated with 100 mg of PE 60 for the time indicated, and supernatant fluids were then assayed for unadsorbed HA activity. virus might behave the same as other viruses in respect to elution from PE. The following experiment was performed to determine the optimal parameters for elution of adsorbed influenza virus from PE 60. The virus stock was treated with ammonium sul-

VOL. 23, 1972 CONCENTRATION OF INFLUENZA VIRUS 743 TABLE 5. Elution of influenza virus from PE 60 HA titer of eluates Control Samples testeda supernatant Distilled Trisfluids buffered wtr (HA titer) water saline Control 1,280 Supernatant fluid < 20 of PE-treated virus 1st water wash at <20 2nd water wash at <20 3rd water wash at <20 Packed PE treated with distilled water or Tris buffer at ph: 7.5 160 20 9.0 320 320 10.0 1,280 320 11.0 1,280 640 12.0 1,280 640 13.0 <20 <20 a PR-8 in undiluted allantoic fluid (200 ml) was treated with ammonium sulfate as described in the footnote to Table 3. Then 500 mg of PE 60 was added and the mixture was stirred for 3 hr at 4 C at ph 5. The suspension was centrifuged at 2,000 rev/min, and the supernatant fluid was assayed for unadsorbed hemagglutination (HA) activity. The packed PE was washed three times with 200 ml of distilled water, and each supernatant washing was assayed. The packed PE was then suspended in water, and a homogenous suspension of 10 ml was distributed to each of 20 test tubes. The tubes were centrifuged at 2,000 rev/min for 5 min, the supernatant fluids were discarded, and the packed PE was treated with 10 ml of distilled water or 10 ml of Trisbuffered saline at the ph levels indicated, all at 4 C. All eluents were tested for ph levels to assure that ph was constant for at least 5 min. NaOH or HCI was used for adjustment of ph levels as necessary. The tubes were then centrifuged in the cold at 2,000 rev/min for 5 min, and supernatant fluids were collected and made neutral (ph 7.0 to 7.2) with HCI and then assayed for HA activity. fate, and PE 60 was added as described above. After repeated washings with distilled water, the resuspended PE-virus complex was distributed into replicate tubes. The packed PE-virus complex was then treated with distilled water or tris(hydroxymethyl)aminomethane (Tris)- buffered saline at various ph levels to determine the optimal eluent. The experimental procedures and results of this test are shown in Table 5. As indicated from the data, influenza virus is completely eluted at ph 10.0 to 12.0 in distilled water. Salt solutions at elevated ph levels also eluted virus, but not completely. Purity of PE virus concentrates. By using the procedure described in the footnote of Table 5, but with a final ph 11.5 eluate of onetenth the original virus fluid volume, a 10 x virus concentrate was prepared. The titer of the control PR-8 virus was 1:640, and that of the PE concentrates was 1:6,400, indicating total recovery of the virus in the concentrate. Nonviral protein was determined by the standard Lowry test (4) and by a new method recently devised in this laboratory (14): the discontinuous counterimmunoelectrophoresis method (DCIE). The experimental procedures and results are shown in Table 6. The Lowry test detected protein (extinction) in untreated PR-8 allantoic fluids up to an eightfold dilution; this was equated with an albumin control to indicate that the undiluted PR-8 allantoic fluid contained 2.5 ug of protein per ml. The 10 x PE concentrate failed to react with the Lowry reagents even when tested in the undiluted state, indicating at least an 80-fold reduction of nonviral protein. TABLE 6. Purity of PE virus concentrates DCIE test (antiserum)c Dilution of virusa L-y - test" Undi- 1:2 1:4 1:8 luted Control, untreated PR-8 allantoic fluids Undiluted + 1:2 + 1:4 + 1:8 + 1:16 0 10-2 ++++ ++++ ++ 4 10-' ++++ + 0O 10-4 ++ + 0 0 10-' 0 0 0 0 PE concentrate Undiluted 0 ± A= 1:2 + + ++ 0 1:4 0 0 + 0 1:8 0 0 0 0 a Two-millimeter wells 1 cm apart in 1% agarose [3 to 3.5 ml per slide (25 by 75 mm)] containing Veronal buffer, ph 8.6, 0.015 gm, were filled with different dilutions of antigens, and opposing wells were filled with different dilutions of antiserum. The agarose slides were bridged with 0.075 jum of buffer (14). Normal baboon serum samples served as a control, and no reactivity occurred; the negative results with the control serum are omitted from the table. "Scoring of Lowry test: + = positive reaction, 0 sample has same absorbancy as control blank. c Scoring of precipitin lines: 0 = no precipitin, to + + + + = very discrete, readily apparent precipitin line (maximum reaction). DCIE, discontinuous counterimmunoelectrophoresis.

744 WALLIS, HOMMA, AND MELNICK APPL. MICROBIOL. A more dramatic indication of the purification by the PE method is illustrated in Table 6, using the DCIE method (14). Control, untreated PR-8 allantoic fluids tested against normal allantoic antiserum (made in baboons) manifested precipitin lines at antigen dilutions up to 1:10,000. On the other hand, the PEpurified concentrate reacted with the antiserum, but only at dilutions up to 1:4. From these results it is evident that a 2,500-fold reduction in precipitable nonviral protein had been removed during the PE purification method. Cellulose acetate strip electrophoresis of allantoic fluids indicated that all detectable proteins were acidic in nature and moved in the direction to react with the antiserum. DISCUSSION The concentration and purification of influenza virus is of signal importance, since the nonviral proteins present in virus harvests obtained from embryonated eggs and used for vaccine production may sensitize persons inoculated with the vaccine. Currently no methods have been described which will simply and economically concentrate and purify influenza virus. The adsorption of influenza virus on and elution off insoluble salts, resin exchanges, erythrocytes, and a variety of earths have been reported (1, 2, 4, 8-12). However, these latter methods are not feasible for use with large volumes of virus fluids and are often dependent upon using partially purified virus to obtain efficient results. Virus stocks must be dialyzed or ultracentrifuged before use with adsorbents to remove interfering salts or organics, or both, which would occupy the reactive sites on the adsorbent and preclude efficient virus concentration. Recently, it has been reported that influenza viruses could be concentrated and purified by zonal centrifugation (7), and such concentrates have been shown to be effective immunizing antigens (6). The laborious and expensive procedures for this type of purification required that the virus be precipitated by barium salts, followed by isopycnic banding in a sucrose gradient with additional steps to remove the contaminating materials used for concentrating the virus. This method yielded a loss of 38% of the total virus initially used. In this current preliminary report, we have shown that influenza virus from whole allantoic fluid can be efficiently and simply concentrated on and eluted off an insoluble polyelectrolyte, with the final virus concentrate manifesting negligible amounts of nonviral protein. Recently, Vonka (personal communication) has confirmed our findings with the PR-8 strain and has shown that the A2 Hong Kong strain of influenza virus behaves in similar fashion. He was able effectively to concentrate the influenza virus with results indicating the high purity that we obtained. ACKNOWLEDGMENT This study was supported by a grant from the Monsanto Company, St. Louis, Mo. LITERATURE CITED 1. Frommhagen, L. H., and C. A. Knight. 1959. Column purification of influenza virus. Virology 8:198-208. 2. Graham, A. F., and L. McClelland. 1950. Uptake of radioactive phosphorus by influenza virus A (PR8 strain). Can. J. Res. 28:121-134. 3. Grinstein, S., J. L. Melnick, and C. Wallis. 1970. Virus isolations from sewage and from a stream receiving effluents of sewage treatment plants. Bull. World Health Organ. 42:291-296. 4. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 5. Miller, H. K., and R. W. Schlesinger. 1955. Differentiation and purification of influenza viruses by adsorption on aluminum phosphate. J. Immunol. 75:155-160. 6. Peck, F. B., Jr. 1968. 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