Factors in the Membrane Filtration of Enteroviruses

Transcription

1 APPLIED MICROBIOLOGY, May, 1965 Copyright ) 1965 American Society for Microbiology Vol. 13, No. 3 Printed in U.S.A. Factors in the Membrane Filtration of Enteroviruses D. 0. CLIVER Food Research Institute and Department of Microbiology, University of Chicago, Chicago, Illinois Received for publication 28 December 1964 ABSTRACT CLIVER, D. 0. (University of Chicago, Chicago, Ill.). Factors in the membrane filtration of enteroviruses. Appl. Microbiol. 13: The filtration of two species of enteroviruses through membranes of porosity ranging from 50 to 220 my was studied. It was shown that extensive or total losses of virus may attend filtration at these porosities, apparently owing to adsorption of the virus to the membrane matrix. This could be minimized by the incorporation of serum into the virus suspension at the time of filtration, or by pretreating the membrane with serum or with a gelatin solution. It was also found that the first few drops of filtrate, even under optimal conditions, were likely to be virus-free, so that the filtration of too small a volume of virus suspension would result in a relatively great loss of titer. The degree to which these factors were critical was found to decrease with increasing pore diameter. In the course of experiments relating to viruses the sizes of simian viruses of several groups, and in foods, it became necessary to devise means of suggested that their techniques might be used for extracting enteroviruses from foods into a fluid preliminary typing of virus isolates. The viruses suspension and of concentrating this suspension filtered were contained in undiluted tissue culture fluid and were found to lose 0.3 to 1.5 logio to insure the detection of such viruses as might be present. Two applications of membrane filtration units of infectivity during clarifying filtrations to this work were envisioned. The first related to with Seitz pads or Millipore GS (220-m, porosity) the clarification of the food extract and the removal of coincident microbial contaminants of virus in Millipore filtrates was more nearly membranes. Their data indicated that recovery while still retaining the virus in the filtrate. The quantitative than that in gradocol filtrates at second related to the development of methods for comparable porosity. concentrating enteroviruses from food extracts. It had become evident in preliminary studies that MATERIALS AND METHODS the virus preparations in use must contain a certain proportion of aggregates. As these aggregates was obtained from the Viral and Rickettsial Reg- Viruses. Poliovirus type 1 (Po-1), strain CHAT, were broken up in the course of manipulations, istry of the American Type Culture Collection. it was not unusual for the post-treatment suspensions to contain a greater number of infectious including four subcultures from isolated plaques, It had been through 15 tissue culture passages, units than had originally been present. For this in HeLa, primary rhesus monkey kidney (PMK), reason, means were sought of filtering virus at WISH, KB, and WI-26 cells. The preparations limiting porosity immediately before use, thus were stored at -20 C as an undiluted harvest eliminating the bulk of the aggregates while (cells and fluid) of infection PMK titering losing little of the input virus titer. In the present plaque-forming units (PFU)/ml, or as a diluted suspension of the above in a maintenance medium studies, the virus preparations employed were titering PFU/ml. Coxsackievirus B-2 (CB-2) produced in tissue culture and were diluted in a was obtained from Dorothy Hamre (Department variety of fluids, but were not extracted from of Medicine, University of Chicago) as a field foods. The findings reported here are general and isolate which had been passed nine times in HEp-2 will be applied subsequently to enteroviruses in cells. It was passed once in WI-26 and eight times food extracts. in PMK, including three subcultures from isolated Black (1958) summarized reports relating the plaques. The preparations employed were stored diameters of virus particles to the limiting pore at -20 C as an undiluted harvest of infected PMK diameters for gradocol filtration. He proposed a titering PFU/ml, or as a diluted suspension of the above in a maintenance medium working figure of 0.64 for the ratio of titering particle PFU/ml. In early passages, this virus was diameter to limiting pore diameter. Atoynatan inhibited by the overlay medium described below, and Hsiung (1964) recently reported the use of and had to be plaqued under the medium described Millipore and gradocol membranes in determining by Wallis, Melnick, and Bianchi (1962). The final 417

2 418 CLIVER APPL. MICROBIOL. preparations were found to yield higher plaque titers under the overlay medium used with Po-1. Tissue cultures. PMK cultures were prepared from the kidneys of rhesus monkeys (Macaca mulatta) by a method described previously (Gibbs and Cliver, Health Lab. Sci., in press). They were maintained until use in a mixture of equal parts of medium 199 and Eagle's minimal essential medium at room temperature. The volume of inoculum was 0.5 ml per flask, and adsorption was allowed to proceed at room temperature for 2 hr with manual rocking at intervals. Agar overlays consisted of 5 ml per flask of Earle's balanced salt solution (EBSS) containing 0.5 g of lactalbumin hydrolysate, 1.25 g of Noble agar, 0.51 g of MgCl2, 1.5 mg of neutral red, 10,000 units of penicillin G, and 10 mg of dihydrostreptomycin sulfate, per 100 ml. Filtration. The membranes employed and their porosities were: Gelman GM-6 (450 m,), GM-8 (200 mis), GM-9 (100 m,u), and GM-10 (50 m,u); Millipore GS (220 m,), VC (100 m,u), VM (50 m,u), and VF (10 m,u); and Schleicher and Schuell B17 (20 to 35 m,u). All Gelman membranes and the Millipore GS membranes could be sterilized either by autoclaving at 121 C for 15 min or by ultraviolet light (UV). The remaining types of membranes were sterilized by UV, with the few exceptions indicated. The holders employed were the Millipore microsyringe filter holder (MH) and the Gelman centrifugal filter holder (CH). These holders accept membranes 25 mm in diameter, and have effective filtering areas of approximately 3.9 cm2. Because of the pressures generated in filtrations using the MH with Multifit (Becton, Dickinson and Co., Rutherford, N.J.) syringes and membranes of 100 m, or smaller porosity, there was back-flow of virus suspension along the syringe plunger. This was prevented by a very light application of high-vacuum silicone stopcock grease to the plunger. Filtration at these porosities was also facilitated by means of a needle and a 20-ml Vacutainer tube (16 X 200 mm). The CH were found to be of no value when used with 100-m,i or smaller porosity membranes in a centrifuge. Three modifications were performed. First, the long (8.7 cm) funnel barrel was fitted with a no. 3 rubber stopper which had been bored and threaded on a length of polypropylene tube with a glass-wool packed bulb blown in it. Second, two flats were filed on the rim of the lower section of the holder to permit application of an open-end wrench to this part while tightening the top collar with a pipe wrench. Finally, the apparatus was mounted on a 50-ml round-bottomed polypropylene centrifuge tube, and the stopper was clamped in place. These modifications permitted filtration with air pressure at 35 psi. The diluents employed in filtration and recovery of viruses included deionized water; EBSS; EBSS plus 0.5% lactalbumin hydrolysate (E-Lac); medium 199; 1 M MgCl2; M phosphate buffers at ph 7.0, 7.5, 8.0, and 8.5, and M phosphate buffers which, when mixed with equal volumes of those just listed, would give a ph of 7.2 (Humason, 1962); and a solution called saline Y, containing 7.14 g of NaCl, g of KCI, g of KH2PO4, g of K2HPO4, g of NaHCO3, and g of phenol red, per 100 ml of final solution in deionized water. To these were added, where indicated, newborn bovine serum (Bo), chicken serum (Ch), or a solution in 100 ml of Hanks' balanced salt solution of 2 g of gelatin (G). The additives and their concentrations are indicated by the above abbreviations and a subscript denoting the percentage of additive (v/v) employed. Thus EBSS Bo2 refers to Earle's balanced salt solution with an added 2% of calf serum. Where possible, the virus titers (in PFU) of the filtrates have been compared directly with those of the same virus suspensions before filtration. The procedural complexities of certain experiments have made it necessary to compare the observations with a "null count." The null count is an estimate, projected from the controls in the same experiment, of the number of plaques which would have been observed if membrane filtration had been totally without effect. RESULTS Preliminary experiments. Po-1 (2 ml) was diluted in 8 ml of medium 199, and was filtered serially through Gelman membranes of 200, 100, and 50 m, in CH on the centrifuge (1,500 X g). The 50-m,A membrane was inverted and backwashed with 9 ml of medium 199 in the same manner. Each of these four filtrates was sampled and titrated by the plaque method. The titers, in PFU/ml, were found to be 2.3 X 107, 8 X 105, <104, and <105, respectively. The rate of filtration appeared to approach zero asymptotically as the weight of the fluid column above the membrane diminished, so no further filtrations were performed on the centrifuge. Then, 1 ml of Po-1 was diluted in 9 ml of medium 199 and filtered serially through Gelman membranes of 200, 100, and 50 m,u in CH modified as described previously and under positive air pressure. Because of volume lost in the filter apparatus and in sampling, the volume of the 50-m, filtrate was only 5 ml. Therefore, this membrane was inverted and back-washed with 5 ml of medium 199. A prefiltration sample and samples of the four filtrates were titrated by the plaque method, and were found to contain 3.1 X 107, 1.6 X 107, 2 X 103, <102, and < 102 PFU/ml, respectively. Next, 1 ml of Po-1 was diluted in 9 ml of medium 199, sampled, and filtered serially through Gelman membranes of 450, 200, 100, and 50 m, in CH under positive air pressure. Samples of the prefiltration mixture and of each of the filtrates were titrated in tubes, and were found to contain 6, 5, 5, <1, and <1 log12 TCD/0.1 ml. At this point it was evident that the porosity of

3 VOL. 13, 1965 MEMBRANE FILTRATION OF ENTEROVIRUSES 419 the mnembrane was not the sole limiting factor in the retention of poliovirus. First, a sharp decrease in titer had been observed with filtration at 100- m,i porosity, more than three times the diameter of the individual virus particle. Second, virus retained by the membrane could not be recovered at detectable levels by passage of medium 199 through the membrane in the reverse direction. A less concentrated suspension of Po-1 (1 ml) was diluted in 4 ml of medium 199 Bo2, sampled, and filtered serially through Gelman membranes of 450, 200, 100, and 50 m,u in CH under air pressure. Each membrane was then inverted and back-washed with 5 ml of 1 M MgCl2. The prefiltration sample was found to contain 4 log10 TcD/0.1 ml, by the tube method. The filtrates contained 4, 5, 4, and 3 logl0 TcD/0.1 ml, respectively. Each of the back-wash fluids contained 3 log1o TCD/0.1 ml. Membrane porosity and serial filtration. The results of the last preliminary experiment described were taken to indicate that, with virus suspended in medium 199 Bo2, the pore size of the membrane was more nearly a limiting factor in retention of Po-1. Therefore, 1 ml of Po-1, diluted in maintenance medium, was added to 4 ml of medium 199 Bo2. The mixture was sampled and filtered serially through Gelman membranes of 450, 200, 100, and 50 m,u in CH under positive air pressure. Samples of the original suspension and of the fluid after each filtration were found to contain 3.1 X 105, 2.6 X 105, 2.6 X 105, 1.4 X 105, and 3.9 X 104 PFU/ml. To determine whether previous membrane filtration of a virus suspension influenced retention by another membrane of the same porosity, another experiment was performed. A suspension of Po-1 in medium 199 Bo2, found to contain 2.4 X 104 PFU/ml, was passed through a Gelman membrane of 100-m,u porosity in CH, under positive air pressure. The filtrate contained 2.3 X 104 PFU/ml. Half the filtrate was passed through another 100-m,M membrane, and was found to contain only 1.5 X 104 PFU/ml after filtration. The other half of the first filtrate was filtered at 50-m, porosity, and was found to contain 1.1 X 104 PFU/ml. After passage through a second 50- m,u membrane, the titer of this suspension was reduced to 7 X 103 PFU/ml. Parallel filtration. A suspension of Po-1 in medium 199 Bo2 was prepared which contained 1.5 X 105 PFU/ml; 3-ml quantities of this were filtered through Gelman membranes of 450, 200, 100, and 50 mu in CH under air pressure. The titers of the filtrates were 2.3 X 105, 1.4 X 105, 1.2 X 101, and 5.3 X 104 PFU/ml, respectively. Except for the observed increase in titer after filtration at 450-m,u porosity, these values do not differ greatly from those observed with serial filtration of Po-1 in medium 199 Bo2. An experiment was performed to test directly the effect of 2% bovine serum in the medium 199 diluent upon the efficiency of filtration by Gelman membranes of 50, 100, and 200 mp. Also included was a test of the influence of the filter holder upon the observed efficiency of filtration. CB-2 virus was diluted in 199 and 199 Bo2 and tested at all possible combinations of the porosities listed above in CH and MH and these diluents. CB-2 (5 ml) at 7.0 X 102 PFU/ml in 199 Bo2 was passed through one membrane of each porosity in each holder. The mean titer of the six filtrates was 4.6 X 102 PFU/ml, with no apparent variation attributable either to porosity or to the holders. CB-2 at 5.6 X 102 PFU/ml in 199 was filtered with a set of membranes and holders like that just described; when these filtrates were tested, no plaques were observed. On the basis of these results, it appeared that bovine serum was necessary for efficient filtration of enterovirus in 199 but the choice of filter holders had little influence. Thereafter, filtrations were done in CH or MH as convenient. Influence of diluent. Since it appeared that medium 199 without serum was an unfavorable diluent for membrane filtration of enteroviruses, a preliminary test was performed to determine whether the same was true of some other diluents without serum. Po-1 was diluted in saline Y to 14 PFU/ml, E-Lac to 12 PFU/ml, 199 to 16 PFU/ ml, and 199 Bo2 to 20 PFU/ml; 3 ml of each suspension were passed through a Gelman 50-m,u membrane in a CH under positive air pressure. The 199 Bo2 filtrate contained 13 PFU/ml, whereas none of the other three was found to contain any. Thus, it appeared that serum, or possibly some other source of protein, was necessary for the efficient passage of enterovirus through a Gelman 50-m, membrane. A preliminary experiment was conducted to test the interaction of medium 199 with various protein sources and of bovine serum with various diluents in membrane filtration. Po-1 was diluted in 199 Bo2, 199 Ch2, 199 G14, E-Lac Bo2 and EBSS Bo2, and deionized water Bo2; 10 ml of each suspension were passed through a Gelman 50-m, membrane in a CH under positive air pressure. The times required to complete these filtrations ranged from 5 to 11 min, except for the 199 G14 suspension which had not been completed when it was stopped after 1.5 hr. The deionized water Bo2 was inoculated initially with twice the volume of Po-1 used with the other diluents, because the filtrate had to be mixed with an equal volume of double-strength diluent before it could be inoculated on the cell sheets. The null count for these

4 420 CLIVER APPL. MICROBIOL. samples was approximately 47 PFU. The counts observed when the filtrates were tested were 46 PFU with 199 Bo2, 63 PFU with 199 Ch2, 0 PFU with 199 G14, 49 PFU with E-Lac Bo2, 53 PFU with EBSS Bo2, and 23 PFU with deionized water Bo2. Although this experiment was only preliminary, there appeared to be sufficient evidence to reject gelatin as a source of protein in the diluent. It also seemed likely that water with bovine serum in it was a less desirable combination than some of the others tested. Another experiment was conducted to determine the relative merit of deionized water without serum as a diluent. In this case, CB-2 was filtered in 10 ml of various diluents through Gelman 200- m,u membranes in MH on syringes. The titers of the filtrates were found to be 51 PFU/ml in deionized water, 0 PFU/ml in 199, and 62 PFU/ml in 199 Bo2. This indicated that deionized water was considerably more effective than 199 for filtration with Gelman 200-mg& membranes. Another experiment was designed to determine the merit of water as a diluent at a variety of membrane porosities. Po-1, which had been filtered at 50 m,, was diluted to 31 PFU/ml in deionized water, which gave a null count of 15.5 PFU/ml after each sample was diluted with an equal volume of double-strength diluent and tested. Two membranes of each porosity were used to filter 5 ml of virus suspension each. The membranes employed were Millipore 10 m,u, Schleicher and Schuell 30 m,u, and Gelman 50 and 100 m,u in CH under positive air pressure, and Gelman 200 and 450 ma in MH on syringes. The plaque counts obtained with the duplicate filtrates were 0 and 0 at 10 m,u, 0 and 0 at 30 m,u, 4 and 0 at 50 m1a, 1 and 0 at 100 m,u, 19 and 14 at 200 m,u, and 17 and 13 at 450 m,u. A similar experiment was performed with CB-2 virus, except that in this case one membrane at each porosity was used to filter virus in water and the other to filter virus in 199 Bo2. The null count was approximately 21.5 PFU in this experiment. The filtrates in water and 199 Bo2 gave plaque counts of 0 and 0 at 10 m,u, 0 and 0 at 30 m,u, 0 and 8 at 50 mya, 0 and 13 at 100 mjp, 20 and 15 at 200 m,u, and 20 and 22 at 450 m,. These results have been taken to indicate that water is a useful diluent for membrane filtration of enteroviruses at porosities of at least 200 my, but is of questionable utility at smaller pore diameters. Since the loss of virus in filtration seemed to be the result of adsorption rather than mechanical retention, it was decided to determine whether successive fractions of filtrate differed in their virus content in a manner analogous to fractions obtained from a chromatographic column. Two Gelman 50-m,u membranes were assembled in MH and attached to 5-ml Cornwall syringes containing Po-1 in 199 Bo2 at 4.1 X 102 PFU/ml. A 19-gauge needle was attached to each MH, and the plunger of the Cornwall syringe was advanced by turning the adjusting screw so that drops could be counted. The 19-gauge needles had been calibrated and found to deliver about ml of 199 Bo2 per drop when held vertically. Three-drop fractions, starting with drops 1, 4, 51, 54, 101, 104, 201, and 204, were collected directly on PMK cell sheets. Drops not being delivered directly into the tissue cultures were collected in a single tube and titrated to provide an estimate of the grand mean titer of the virus in the filtrates. This was found to be 3.7 X 102 PFU/ml. The results of the dropwise determinations are presented in Fig. 1. The total volumes of filtrate represented are about 3.3 ml for each filtration because of the volume of fluid required to fill the MH. The fluid present in the chamber above the membrane was eventually filtered by drawing air into the syringe and applying mechanical force, but this fluid was not collected in dropwise fashion. It was, however, represented in the tube used for determining the mean titer of the filtrates. Two features of the curves shown in Fig. 1 were deemed significant. First, virus could not be detected in at least the first six drops collected in each filtration. Second, the levels of virus in late fractions of the first filtration were well above the mean titer of the filtrates and might not yet have reached their maximum. For purposes of comparison it can be estimated on the basis of the mean titer observed and a volume of ml for each three-drop fraction that the mean plaque count for all such fractions was about 18. IL a o o I D R O PS FIG. 1. Replicate filtrations of Po-1 virus in 199 Bo2 through Gelman 5O-mA membranes. Each point represents the PFU content of a three-drop sample (ca ml) taken directly into a PMK at the indicated stage in the filtration.

5 VOL. 13, 1965 MEMBRANE FILTRATION OF ENTEROVIRUSES 421 It was desired to learn more Iprecisely the each case were evidently low in titer, were colshapes of the curves at their extremi.es and to de- lected directly in tissue cultures rather than in termine the influences of diluent arid virus con- the tubes from which the cumulative samples centration upon these observationis. To these were taken. On the other hand, the results of ends, four suspensions of Po-1 we,re prepared. both of these experiments indicate that filtration These comprised Po-1 at approximlately 5.3 X of volumes of virus suspension less than 3 ml, 102 PFU/ml in water, 199, and 199) Bo2, and at even when a favorable diluent such as 199 Bo2 1.3 X 102 PFU/ml in 199 Bo2. Eac' h of the first is employed, inserts a strong negative bias into three suspensions was filtered throutgh two mem- the system. branes by the method employed in the previous It had been demonstrated previously that both experiment, but the portion of each ifiltrate which chicken and bovine serum were of value in was not inoculated directly into cultures was diluents for membrane filtration of enteroviruses. collected separately and titrated. The fourth To test the relative merits of these sera, Po-1 mixture was filtered in the same fashion, but was diluted in E-Lac with 0.5% and 2% of each 'from a 10-ml Cornwall syringe. Sirice the virus serum; 5 ml of each suspension were filtered concentration was one-fourth as E4reat in this through a Gelman 100-m, membrane in CH and suspension as in the others, fractionls of 12 drops a Millipore 100-m, membrane in CH under rather than 3 were collected. Foor simplicity, positive air pressure, with one exception. Be- (Fig. 2) cause of some leakage from the holder used in approximate curves have been draiwn to summarize the results of each I)air of filtra- filtering the E-Lac Ch2 suspension through the tions. This is justified by the similarrities in mean Gelman membrane, only 3 ml of this suspension titers determined for correspondiing filtrates. were filtered. The results of this experiment are These were 8 X 101 and 8 X 101 ffor the water presented in Table 1. Because of what has been suspension, <101 and <101 for the 199 sus- learned about the' effect of volume upon the pension, 5.0 X 102 and 5.2 X 102 for the first apparent efficiency of filtration, it seems likely 199 Bo2 suspension, and 1.5 X 102 and 1.6 X 102 that a portion of the decrease in efficiency obfor the second 199 Bo2 suspension. From this it served with the single 3-ml filtration may have was concluded that the differen ces between come from this source. The most noteworthy replicate points were mainly due to sampling aspect of these data was the apparently diminerror. It should be noted that the lae3t two filtrate ished effect of both sera at 0.5% concentration titers quoted were the only ones hig,her than the in the case of the Millipore 100-m,u membranes. corresponding input titer. This maby have been There appeared to have been no substantive due in part to bias resulting from tbie fact that a difference in efficiency which could be attributed larger proportion of the early dro LL a. 30 i4 - _ * N- x. 20 / I / 10 1 I / 3N, I' 0 * o o---- o2_ n ofe Onto Z(In u Iuu *Juu Juu D R O P S - ps, which in to the species of serum employed. Another experiment was performed to explore the effect of varying concentrations of serum upon the efficiencies of filtration with Gelman - and Millipore membranes. CB-2 at 1.4 X 102 PFU/ml was filtered through 50-mI, membranes in CH, with E-Lac and chicken serum (from 0 to 5 %) as the diluent; 5 ml of each suspension were passed through each membrane under positive air pressure. The times required for all filtrations through Gelman membranes, and for the virus suspension without serum through a Millipore membrane, fell in the range of 10 to 20 min. Approximately 1 hr was required to filter 5 ml of each of the suspensions containing chicken serum through Millipore membranes. The proportions of virus present in the various filtrates ur virus sus- are shown in Fig. 3. It appeared that the low FIG. 2. Dropwise filtration of foi pensions through Gelman 50-m,u mern,lbranes. Each dose-response effect reported for Millipore 100- point represents the mean of determiinations from mj.a membranes (Table 1) was even more protwo replicate filtrations. (1) Po-I in w)ater, 3 drops! nounced in the case of 50-m, membranes. sample; (2) Po-1 in medium 199, 3 X (3) Po-1 in 199 Bo2, 3 drops/sample, r(4) Po-l, at It seemed probable that relatively high concenito 3, in 199 trations of serum in the diluent would be re- one-fourth the concentration used in Bo2, 12 drops/sample. quired if a high proportion of enterovirus was

6 422 CLIVER Bovine * Chicken APPL. MICROBIOL. TABLE 1. Proportions of Po-1 virus pas sing through final mixture would be at ph 7.2. With both 1OO-m,g membranes as a function of species of Gelman 100-m,u and Millipore 100-m,i membranes serum at two concentrations in E-Lcxc diluent in NH with syringes and Vacutainers, no virus Serum Menmbrane could be found in the filtrates of suspensions without serum. Suspensions at ph 7.0 with 2% Species Concn Gelman GM-9 Millipore VC chicken serum were found to contain 71 PFU/ml - after passing a Gelman 100-m, membrane, and 61 PFU/ml after filtration through a Millipore m,u membrane Virus concentration. The results of a number of 0.27 experiments above were compared, even though 1.00 the concentrations of virus in the suspensions to be filtered varied among experiments. The im- * Ratio of titer of filtrate to co: rresponding plicit assumption that the observed efficiency n membrane of filtration is not materially a function of the control. The volume filtered was 5 ml i: except the E-Lac Ch2 with the Gelmar (in which 3 ml were filtered). Lii Hr- 1.0 _ concentration of virus employed is justified to some extent by the results shown in Fig. 2. However, the experiment cited included only a fourfold difference in virus concentration, and titers in other experiments have varied over a much 'G - wider range. Po-1 was diluted in 199 Bo2 and filtered through Gelman 50-miu membranes in CH under positive air pressure at dilutions of 10-4i, 10-5, 10-6, and Where necessary, the filtrates were further diluted to a final level of 10-7, and were tested together with an unfiltered -j LL z 0.8 (I) D 0.6,0 LL 0.4- / 0 sample of virus at The estimated proportions of virus in each filtrate referred to the z I/ / unfiltered sample were 0.38, 0.38, 0.38, and 0.54, respectively. No obvious explanation of the slightly higher return from filtration at ) 0~ M I l l-. dilution is seen, but it is to be noted that all of CL these efficiencies are somewhat low in comparison % SERUM N DILUENT with other experiments. In any case, the variation of virus concentration over a 1,000-fold FIG. 3. Proportions of CB-2 virus pas, sing through range appears to have a relatively minor in- Gelman and Millipore 5O-mM membrane,s as a func- fluence when compared with some of the other tion of the concentration of chicken seruw mn the E-Lac diluent. G, Gelman membrane pmese irhn factors involved in efficiency. ; M, Milli- Method pore membranes. of sterilization. A preliminary test had indicated that there was little difference in the performance of Gelman membranes which had to be gotten through a Millipore mlembrane of been sterilized by autoclaving or by UV. Milli- 50 or 100 m,u, and that these levels of s,erum would pore membranes of 100 m,a or smaller porosity seriously retard filtration. Since each of the early are said not to withstand autoclaving by their experiments relating to the effects of diluents manufacturer, but no information is given reh Gelman garding consequences. had been performed only wit] membranes, it was hoped that some further ex- These questions were investigated with Gelient system man and Millipore 50-mi/ membranes in MH ploration would bring to light a dill; which would function well with bolth types of with syringes and Vacutainers. One of each type membranes. Po-1, which had been fi] Itered at 50 of membrane had been autoclaved, while the m,u, was diluted to a level of 70 PFU/ml in other had been sterilized with UV. All four M phosphate buffer at ph 7.0, 77.5, 8.0, and membranes were used to filter 5 ml of Po-1 at 8.5 (Humason, 1962) and in i phosphate 37.5 PFU/ml in E-Lac Ch2. The proportions of buffer at ph 7.0 with 2% chicken serum added. virus found in the Millipore filtrates were 0 and Since some of these solutions were iinjurious to 0.28 for the UV-sterilized and autoclaved cultured cells, particularly at or abcwve ph 8.0, membranes, with filtration times of 90 and 60 each filtrate was mixed before testi ng with an min, respectively. The corresponding levels of equal volume of phosphate buffer such that the virus in the Gelman filtrates were 0.89 and 1.04,

7 VOL. 13, 1965 MEMBRANE FILTRATION OF ENTEROVIRUSES 423 with each filtration requiring 10 min. This suggested that the Millipore membrane had been materially altered by autoclaving but that there was no great difference in the performance of the autoclaved Gelman membrane. An attempt was made to determine the extent of degradation of the Millipore membrane in autoclaving while verifying the findings with respect to the Gelman membrane. CB-2 was diluted in EBSS Ch2 to 54.5 PFU/ml and in EBSS to 59.5 PFU/ml; 5-ml volumes of the EBSS Ch2 suspension were filtered through a set of membranes identical with those used in the last experiment. Proportions of the virus found in filtrates of UV-sterilized and autoclaved Millipore membranes were 0.12 and 0.29, whereas those for the corresponding Gelman filtrates were 0.95 and 1.05, respectively. The times of filtration were 70, 90, 10, and 10 min. An additional pair of Millipore 50-mg, membranes was used to filter the EBSS suspension. The filtration of 5 ml was completed in 10 min in each case; but, whether the membrane had been autoclaved or sterilized with UV, no virus was found in the filtrate. Thus, it appeared that whatever degradation of the Millipore membranes had been caused by autoclaving, it had not obviated the requirement for serum in filtration. The difference in values for the Gelman membranes in the two experiments was possibly real, but certainly minor. Pretreatment of membranes. It had been shown that serum was necessary for efficient filtration of enteroviruses through both the Gelman and Millipore membranes, with the exception of virus in deionized water at porosities of 200 m,u or larger. However, it was particularly evident with Millipore 50-my membranes that the incorporation of serum into the diluent led to a drastic decrease in the rate of filtration. For these reasons, means were sought whereby the protein from serum or some other source could be used to treat the membranes prior to the virus filtration and accomplish the same purpose. Two Gelman 50-mgu membranes were assembled into CH. One was used to filter 2 ml of bovine serum, and the other was used to filter 5 ml of EBSS Bo2. These were transferred to MH, and were inverted in the process because the membrane pretreated with bovine serum had clogged somewhat. Po-1 (5 ml) at 40.5 PFU/ml in EBSS Bo2 was filtered in a MH with syringe and Vacutainer through a Gelman 50-m,u membrane which had not been pretreated. The time of filtration was 5 min, and the filtrate contained 40 PFU/ml. Po-1 (5 ml) at 40 PFU/ml in EBSS was filtered in the same manner through an untreated membrane, a membrane pretreated with calf serum, and a membrane pretreated with EBSS Bo2. The times of filtration were 5, 30, and 5 min; the filtrates were found to contain 0, 17.5, and 9 PFU/ml, respectively. Thus it appeared that pretreatment of the membrane itself with serum had some merit, but that the method employed had caused some clogging of the membrane which could not be eliminated simply by performing the virus filtration in the opposite direction. An attempt was made to accomplish the same result by soaking the membranes rather than forcing the pretreatment fluids through them. Gelman 200-mg, and Millipore 220-m, membranes were prepared in MH after no pretreatment, 10 min of soaking in chicken serum, and 10 min of soaking in 2% gelatin solution. Each of these was used with a syringe to filter 5 ml of EBSS containing about 42.5 PFU/ml of CB-2. Filtrates from Millipore membranes which had no pretreatment, and which were pretreated with chicken serum and gelatin, were found to contain 0, 42, and 32 PFU/ml, respectively. The corresponding Gelman filtrates contained 0, 21.5, and 36.5 PFU/ml. It seemed advisable to determine whether longer periods of pretreatment would prove advantageous, whether pretreatment by soaking was applicable to membranes of smaller pore size, and whether the coating substance was removed in the course of filtration. Duplicate Gelman 50- and 220-m, membranes and Millipore 50- and 200-m, membranes, all sterilized with UV, were soaked overnight in 2% gelatin at 5 C and assembled into MH. Po-1 was diluted in EBSS and deionized water, the latter suspension having double the virus content of the former to permit dilution of the filtrate in doublestrength medium before testing. The filtrates were collected in 1-ml fractions, and the oddnumbered fractions were discarded. The evennumbered fractions were titrated (Fig. 4). Because of the dead space in the MH chambers above and below the filter membrane, it was more difficult to obtain reproducible samples this way than by the drop method. If each of the filtrates had been collected entirely in one tube and then titrated, it would have been seen that deionized water was not a desirable diluent for use with gelatin-treated membranes. Because of the shapes of the pertinent curves, however, it is further surmised that the effect of the pretreatment diminishes with increasing volumes of water filtered, perhaps because the gelatin is eluted by the water. The shapes of the curves for filtration with EBSS diluent do not differ greatly from those shown in Fig. 1 and Fig. 2, and it seems likely that the coating effect of the gelatin was

8 Z. Q- 30% A 20[ 10 0 *. --" 9-"I 424 CLIVER 1.\ 0 \? s0 -N X--- X_ M ILLI LITERS FIG. 4. Filtrations of Po-1 virus through membranes pretreated with gelatin. (A) Porosity, 50 mg; (B) porosity, 200 to 220 m,u; (1) Gelman membranes, virus in EBSS; (2) Gelman membranes, virus in water; (3) Millipore membranes, virus in EBSS; (4) Millipore membranes, virus in water. not diminished while these filtrations were being performed. A similar set of filtrations was performed with CB-2 virus in EBSS and deionized water, with Gelman and Millipore 100-mA membranes which had been soaked overnight in chicken serum. In this experiment, five 2-ml fractions were collected in the course of each filtration and titrated separately. When the values obtained were plotted in the same way as those in Fig. 4, the curve determined by each set of points could be represented as a straight line of zero slope. Therefore, the values for each filtration were simply averaged and compared with the concentrations of virus in the starting suspensions. The proportions of virus which passed the Millipore membranes pretreated with chicken serum were found to be 0.99 in EBSS and 0.94 in deionized water. The corresponding values for the Gelman membranes were 0.91 and Thus, it appeared that deionized water was not likely to prove a favorable diluent for virus being filtered through pretreated membranes of any of these porosities, with the apparent exception of Millipore membranes pretreated with chicken serum. Isotonic salt solutions seemed to serve well as diluents, and the flow rate through the Millipore membranes was not diminished as it was when serum was incorporated into the virus suspension. DISCUSSION No evidence was obtained to indicate that there is any qualitative difference between the behavior of Po-1 and CB-2 in membrane filtration. It remains to be seen whether the findings of these studies are equally applicable to every other species of enterovirus. -. APPL. MICROBIOL. What must be considered the central theme of these findings is that the nominal pore size of a filter membrane, however carefully this may have been controlled in manufacture, is only conditionally a factor in determining the size of a virus particle that will pass the membrane. Loss of titer in filtration has been reported by Atoynatan and Hsiung (1964), whereas the reports summarized by Black (1958) have generally considered only whether any infectivity at all could be demonstrated in filtered virus suspensions. The results of certain experiments in the present study indicate that, where membrane filtration is used to estimate the particle size of a virus species, injudicious choice of the diluent employed could lead to the conclusion that the diameter of the enterovirus particle is equal to or greater than 200 mp. Diluents which might have led to such an error included medium 199, EBSS, and E-Lac, each of which is in common use in virus research. A major factor in the loss of virus titer in filtration appeared to have been adsorption to the membrane. Adsorption which had taken place with virus in 199 was not reversible by passing 199 through the membrane in the opposite direction, but a portion of the adsorbed virus could be eluted if 1 M MgCl2 was used instead. When passage of virus through a membrane with minimal loss of titer was the desired net result, means of preventing rather than reversing adsorption were sought. With Gelman membranes, it was found that relatively little loss of titer occurred when filtering enterovirus suspensions containing 2% bovine or chicken serum in various media or salt solutions. The species of sera employed were determined solely by what was at hand, so it is entirely possible that others would serve as well. On the other hand, the concentrations of serum required for efficient filtration of enteroviruses through Millipore membranes, particularly at porosities of 50 and 100 m,u, were considerably higher. Further, the presence of serum in diluents for Millipore filtration at these porosities had an extremely adverse effect upon flow rate. The alternative approach which proved fruitful consisted of soaking the membranes overnight in serum or in a 2% gelatin solution before use. This system has not been explored as extensively as that described previously, but only water among the virus diluents tested appears to be capable of removing whatever coating has been left by the treatment. The differences observed in the behavior of the Gelman and Millipore membranes are presumably due to differences in chemical composition. The Gelman membranes are said to consist of

9 VoL. 13, 1965 MEMBRANE FILTRATION OF ENTEROVIRUSES 425 cellulose acetate, whereas the Millipore membranes are formulated primarily of cellulose nitrate. The Millipore membranes of 100 m,u or smaller are said not to withstand autoclaving. It has been shown that sterilization at 121 C for 15 min resulted in only a partial degradation of these membranes; but, since the exact nature of the induced changes could not be demonstrated, the matter was not explored further. When filtrates were collected in fractions, it became evident that the effluents even under ideal conditions were not homogeneous. It is tempting to consider membrane filtration as a microchromatographic process, but this would be an oversimplification. A detailed investigation of the physics of membrane filtration of enteroviruses is beyond the scope of the present study. However, the observations reported would appear to have been the net result of the interactions of several processes, and among these were probably the adsorption and elution of virus from the matrix surfaces, competition of macromolecules for adsorption sites, dissociation of virus aggregates at the membrane surface, and gradual clogging of the pores. The method of filtration employing the Gelman 50-m,u membrane and at least 3 ml of enterovirus suspended in medium with 2% serum has been used as a preliminary to some 44 experiments concerning other subjects. The virus concentrations have covered a range of 103 and the mean loss of titer in these filtrations was approximately 12%. The corresponding losses in limited numbers of filtrations with membranes of larger pore diameter appear to be somewhat less. ACKNOWLEDGMENTS The technical assistance of John Yeatman and Mrs. Jimmie M. Martin is gratefully acknowledged. This investigation was supported by Public Health Service research grant EF from the Division of Environmental Engineering and Food Protection. LITERATURE CITED ATOYNATAN, T., AND G. D. HsiUNG Ultrafiltration of simian viruses. Proc. Soc. Exptl. Biol. Med. 116: BLACK, F. L Relationship between virus particle size and filterability through gradacol membranes. Virology 5: HUMASON, G. L Animal tissue techniques, p W. H. Freeman and Co., San Francisco. WALLIS, C., J. L. MELNICK, AND M. BIANCHI Factors influencing enterovirus and reovirus growth and plaque formation. Texas Rept. Biol. Med. 20: Downloaded from on November 5, 2018 by guest