Ultracentrifugation in the Concentration and Detection

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1 APPLIED MICROBIOLOGY, May, 95 Copyright 95 American Society for Microbiology Vol. 3, No. 3 Printed in U.S.A. Ultracentrifugation in the Concentration and Detection of Enteroviruses DEAN 0. CLIVER AND JOHN YEATMAN Food Research Institute and Department of Microbiology, University of Chicago, Chicago, Illinois Received for publication 7 December 9 ABSTRACT CLIVER, DEAN 0. (University of Chicago, Chicago, Ill.), AND JOHN YEATMAN. Ultracentrifugation in the concentration and detection of enteroviruses. Appl. Microbiol. 3: Ultracentrifugation has been evaluated as a method of concentrating enteroviruses from suspensions whose initial titers ranged from.7 X 0 to. X 0- plaque-forming units (PFU) per ml. A technique employing a "trap" of 0. ml of % gelatin solution at the point at which the pellet forms in tubes for the number 30 and number 50 rotors of the Spinco model L preparative ultracentrifuge has been tested and found to have a number of advantages. Qualitative studies have been performed to determine the sensitivity of the ultracentrifuge technique in detecting the presence of enteroviruses in very dilute suspensions. There was found to be at least a 50% probability of detecting virus present initially at levels as low as 0. PFU per ml by means of the number 50 rotor. The input level for similar results with the number 30 rotor was found to be 0.05 PFU per ml. In a recent review relating to the incidence of viruses in foods, Berg (9) pointed out that there is a lack of available methods for demonstrating the presence of these agents in foods. Poliovirus has recently been recovered from oysters kept in an experimentally contaminated environment (Hedstrm and Lycke, 9). The method employed for isolating virus from the tissues consisted of trituration, ultrasonic treatment, and centrifugal clarification of the suspension. Since no effort was made to concentrate the virus in the tissue homogenates or in samples of the environmental water, the threshold level of virus which was detectable was necessarily rather high. A rationale for testing foods for viruses has been stated previously (Gibbs and Cliver, Health Lab Sci., in press). Work with suspensions of reovirus at very low concentrations has indicated that ultracentrifugation can materially increase the likelihood of demonstrating the presence of this agent. Since the enteroviruses are smaller and therefore slower to sediment, means were sought whereby enteroviruses could be concentrated efficiently in a preparative ultracentrifuge. The present study deals with the concentration of laboratory strains of enteroviruses diluted before ultracentrifugation to various levels in standard diluent. It is expected that these findings will be valid when applied to food extracts and to a variety of other suspensions which may contain virus. Baron (957) studied an analogous problem relating to the detection of residual live poliovirus in formalin-killed vaccines, and recommended the addition of gelatin to a final concentration of 0.0% in the input virus suspension to induce the formation of a relatively firm pellet from which the virus is not dislodged during deceleration of the rotor. The method has since been applied successfully to the detection of enteroviruses in sewage (Gravelle and Chin, 9) and in stool suspensions (Peizer, Mandel, and Weissman, 9). An adaptation of gelatin in the ultracentrifugal concentration of reovirus has been reported (Gibbs and Cliver, in press). This method was tested at high and low input titers of enterovirus. MATERIALS AND METHODS Viruses. Poliovirus type (), strain CHAT, was obtained from the Viral and Rickettsial Registry of the American Type Culture Collection. Since arriving in the laboratory, it had been through 5 tissue culture passages, including four subcultures from isolated plaques in HeLa, primary rhesus monkey kidney (PMK), WISH, KB, and WI- cells. The preparations were stored at -0 C as an undiluted harvest (cells and fluid) of infected PMK titering 0 3 plaque-forming units (PFU) per ml or as a suspension diluted in Earle's balanced salt solution plus 0.5% lactalbumin hydrolysate (E-Lac) and % calf serum containing 0 7PFU per ml. Coxsackievirus B- () was obtained from 37

2 3 CLIVER AND YEATMAN APPL. MICROBIOL. Dorothy Hamre (Department of Medicine, University of Chicago) as a field-isolated strain which had been passed nine times in HEp- cells. It was passed once in WI- and eight times in PMK, including three subcultures from isolated plaques. The preparation employed was stored at -0 C as an undiluted harvest (cells and fluid) of infected PMK titering 07 PFU per ml. In early passages, this virus was inhibited by the overlay medium described below and had to be plaqued under the medium described by Wallis, Melnick, and Bianchi (9). The final preparation was found to yield higher plaque titers under the overlay medium used routinely with. Experience had indicated that these virus preparations occasionally show an anomalous rise in titer during manipulation; this was taken to indicate the presence of a certain proportion of aggregates in suspension. To forestall this, and to render the procedures more nearly quantitative, the virus suspension was passed through a filter membrane at the time of use. The usual procedure involved the dilution of the virus in medium 99 plus %7o "calf serum and filtration through a Gelman GS-0 membrane (50-m, porosity), followed by final dilution to the chosen level in E-Lac diluent. Where filtrations of virus at high titer, such as undiluted tissue-culture suspensions, were being performed, membranes of 00- or 00-m,u porosity were sometimes employed. Cells. PMK cultures were prepared from the kidneys of rhesus monkeys (Macaca mulatta) by a method described previously (Gibbs and Cliver, in press). They were maintained until use in a mixture,of equal parts of medium 99 and Eagle's minimal essential medium at room temperature. Despite some reports indicating otherwise (Kelly and Sanderson, 9; Godtfredsen and von Magnus, 959), it was decided on the weight of evidence that the plaque technique in PMK was a method generally applicable to the detection as well as quantitation of enteroviruses (Hsiung and Melnick, 955; Hsiung, 959). Tests indicated that, for monolayers in plastic tissue-culture flasks, optimal sensitivity was achieved by the use of a 0.5- ml inoculum, an adsorption period of 0 min at room temperature (about 3 C) with periodic manual agitation, and an overlay volume of 5 ml. Although some increase in sensitivity might have been obtained by withholding neutral red from the first agar overlay and adding it at a later time (Darnell, Lockart, and Sawyer, 95), it was decided that a method which was later to be applied to unknowns that might require subculture from plaques had better not require the addition of anything after the first overlay (Mosley and Enders, 9). The agar medium employed consisted of E- Lac with.5 g of Noble agar, 0.5 g of MgCl (Wallis et al., 9),.5 mg of neutral red, 0,000 units of penicillin G, and 0 mg of dihydrostreptomycin sulfate, per 00 ml. Ultracentrifugation. The ultracentrifuge employed was a Spinco model L. Both the number 30 and number 50 rotors, with their corresponding thin-wall polyallomer tubes and aluminum tube caps, were used. The nominal capacity of tubes for the number 30 rotor is 3.5 ml, which at tubes per rotor load gives a total capacity of ml. Tubes for the number 50 rotor are said to accommodate 0 ml each, or a total of 00 ml per rotor load. Experience has indicated that these figures are rather optimistic. When charging the tubes through the small ports provided in the caps, a quantity of air is always trapped which cannot readily be eliminated. Capacity for tubes of the number 30 rotor was found to be about 3 ml each, and for tubes of the number 50 rotor about 9.7 ml each. The levels of virus employed in the various experiments were arbitrarily designated low, medium, and high, with the medium range encompassing titers from 03 to 07 PFU per ml. The serum incorporated into the virus suspension at the time of membrane filtration was diluted to levels of 0.0% or below in preparing the low and medium titer suspensions. The number 30 rotor was run at 30,000 rev/min (maximum, 05,5 X g), and the number 50 rotor at 59,000 rev/ min (maximum, 9,5 X g). With the exceptions noted, a trap consisting of 0. ml of % gelatin solution was applied to the inside of the tube at the point farthest from the axis of rotation, and was collected, after discarding the supernatant fluid, by adding 0. ml of E-Lac diluent at 37 C. The trap and diluent, or diluent alone where no trap was employed, were transferred directly from the tube to a single PMK monolayer by means of a bent Pasteur pipette. At times the residual supernatant fluid which clung to the walls of the tube was removed with a cotton swab before the trap was collected. Where input levels of virus exceeded 3 PFU per tube, the collected precipitate was usually diluted to appropriate levels for plaque titration. The electronic brake of the ultracentrifuge was employed in all runs. RESULTS The first experiments were performed to establish the mechanics of harvesting from ultracentrifuge tubes with and without traps and the applicability of the trap technique at intermediate levels of virus input. A suspension of filtered at 00 mp, was used to charge 0 tubes (five of which contained traps) for the number 50 rotor. The input virus level was 3.5 X 05 PFU per tube, and the time of the run was 0 min. The results are presented as experiment in Table. Fluid ( ml) was left in the bottoms of the untrapped tubes so as not to dislodge the pellet, which was not visible. Since Baron (957) reported that a considerable portion of the virus sedimented in regular ultracentrifugation was to be found in the fluid immediately above the pellet, this may explain the similarity in results with the two methods. In the second experiment, about.3 X 07 PFU of which had been

3 VOL. 3, 95 CONCENTRATION AND DETECTION OF ENTEROVIRUSES Expt TABLE. Method Preliminary experiments with the trap technique for concentrating poliovirus in the number 50 rotor Supernatant fluid Precipitate Volurne Titer Total Volume Titer Total (mi/tube) (PFU/ml) (PFU/tube) Input (ml/tube) (PFU/ml) (PFU/tube) Input No trap X 03. X X 05.7 X 05 7 Trap 9..0 X X X 0. X 05 0 No trap 9..0 X 0 9. X X X 0 5 Trap X 0 3. X X X 0 5 TABLE. Ultracentrifugation of high-titer suspensions of poliovirus by the trap method Supernatant fluid Precipitate Expt ExptRotor Roo Tim Time of of (PF/ Input- tube) Titer Total _-_ run (PFU/tube) Titer TotalTie Toa hr (PFU/ml) PF Tubel Input Ilu (PFU/m) (PF /tube) input (PFU/ml) (PFU/tube) Input X 07 l0 X 05 l0 X 0i 3 3. X 0 3. X X X X X 00. X X X 0.3 X0 5.0 X X 09 filtered at 00 m,u were placed in each of four tubes (two of which contained traps) for the number 50 rotor, and were run for 0 min. The supernatant fluids in the untrapped tubes were taken off completely (Table ). Attempts were also made, by use of the trap method, to determine how completely virus was sedimented from medium-titer suspensions by centrifugation in the number 50 rotor for 0, 90, and 0 min. Although the quantity of virus recoverable from the traps approached 00% of the input by 90 min, 0.% could still be found in the supernatant liquid after 0 min. No virus was recovered in the supernatant fluid after 0 min, when the input titer was below 0 PFU per ml, so this was the time selected for ultracentrifugation of low-titer suspensions. With the number 30 rotor, similar results could be achieved by centrifugation for 5 hr. High-titer virus suspensions. An attempt was made to apply the trap method with the number 50 rotor directly to virus harvested from tissue culture. Enough was thawed to permit filling two tubes without dilution. This was filtered first at 00 m,u and then at 50 m,. The maintenance medium for the cells in which the virus has been propagated was medium 99, which has since been found to prevent from passing efficiently through a 50-m,u membrane. As a result, N 39 the input per tube was only 3. X 07 PFU. The results are presented as experiment in Table. There was no obvious reason why so much of the virus should still have remained in the supernatant fluid after hr, but it was conjectured that perhaps the virus itself was establishing a density gradient which retarded the sedimentation of the last of the virus. To overcome this, if possible, the time of the next ultracentrifugation was increased to hr. The undiluted suspension was filtered only at 00 m,u, to minimize loss at that stage. These results are presented as experiment in Table. It can be seen that a larger portion of the virus could be sedimented by prolonging the ultracentrifugation, but that a finite quantity would still remain in the supernatant fluid. The increase of yield over input was attributed to aggregates of virus passing the 00-m,u filter and later dissociating. An attempt was made to apply a similar procedure to the number 30 rotor. In this case the undiluted suspension was extracted with chloroform, to denature cell fragments to which virus might be aggregated, and was filtered at 00 mu. These results appear as experiment 3 in Table. Low titers with and without traps. Efforts were made to determine with each rotor whether the method involving the use of the gelatin trap was significantly more efficient in detecting small numbers of infectious units. Most of these experiments involved direct comparisons of two or more methods in the same rotor during the same run. The order in which tubes were charged was generally adjusted so as to cancel any possible bias. All virus employed was filtered at 50-m,A porosity at the beginning of each experiment.

4 390 CLIVER AND YEATMAN APPL. MICROBIOL. TABLE 3. Comparisons of methods for concentrating enteroviruses from low-titer suspensions in the number 30 rotor. Virus 3 5 Po-l 3 Input (PFU/ vs. vs. vs. vs. vs. 3 vs. 3 vs. vs. vs. vs. vs. vs. Method of No. tubes Recovery Methods (PFU/ compared Significance of difference (P) None None <0.0 <0.00 <0.0 The methods employed were assigned numbers on an arbitrary basis. Method consisted of ultracentrifugation with no traps. Method was the trap technique which has been described. Method 3 was a variation on the trap technique in tubes for the number 30 rotor in which 0.5 ml of % gelatin solution was applied along the side of the tube farthest from the axis of rotation, to extend from the skirt of the cap to the bottom of the tube. Method consisted of incorporating 0.0% gelatin into the input virus suspension, in lieu of a trap, as suggested by Baron (957). Tests for significance of differences among methods were performed with a t test modified for small sample sizes (Dixon and Massey, 957). The results of experiments with these methods and the number 30 rotor are reported in Table 3. The levels of virus employed were increased after the third experiment because it had become evident that significant differences would be hard to obtain with as few PFU as had been used previously. Even so, the variation within groups, as in experiment 5, was sometimes great enough to prevent differences from being significant. Method could not be shown to be superior to method in the number 30 rotor. Results of similar experiments performed with the number 50 rotor are presented in Table. Experiments la and lb were performed on different days; although they were done so as to make the results comparable, the difference in input titers obtained for the two days made it necessary to compare the yields as percentages of the inputs. Application of the trap method to qualitative levels of virus input. Although the trap method could not be shown statistically to be consistently more efficient than simple ultracentrifugation in concentrating virus from dilute suspensions, its sensitivity did appear to be equal to or better than those of the other methods tested. Therefore, the trap technique was chosen for experiments with suspensions containing less than PFU per ml, at which levels there is a significant probability that the presence of virus would not have been detected if some method of concentration had not been employed. The input titers of virus actually determined for the various experiments ranged from 0.0 to 0.55 PFU per ml. For the purpose to which this procedure was to be put, the fundamental concern is with the probability of detecting viius which is present at levels in this range. It is useful to define a variable, p(+), which may be used to estimate the probability of a positive finding in testing a sample having a particular virus titer. One defines p(+) = - p(o), where p(o) is the probability of detecting no virus predicted by the Poisson formula: p(w) = e-nnw/w!. The variable W in TABLE. Comparisons of methods for concentrating enteroviruses from low-titer suspensions in the number 50 rotor K la lb 3 virus 0 ) 0.0 v: o +, Z Input ee F er Methods (PFU/ compared vs. * vs. vs. vs. vs. vs. vs. vs. this context is defined as the number of plaques observed, and n in the number of PFU per unit of inoculum. The probability of a negative test (W = 0) may then be estimated from: p(0) = e. The unit of inoculum for the initial virus suspension was arbitrarily set at ml. The comparable statistic for samples after ultracentrifu- IptRecov- Significance of difference (P) <0.0 * This comparison was performed with the data for recovered PFU recalculated as percentages of input PFU.

5 VOL. 3, 95 CONCENTRATION AND) DETECTION OF ENTEROVIRUSES 39 TABLE 5. Detection of enteroviruses at very low initial titers by means of ultracentrifugal concentration Rotor No. of tubes Virus Po-l Input concn (PFU/ ml) P(+) gation, pobs, was defined as the actual proportion of traps in an experiment from which at least PFU was recovered. An additional figure, r, defined as the ratio of PFU recovered to PFU put into the tube, was computed for each experiment. Because of the very low initial levels of virus being employed, no attempt was made to detect residual virus in the supernatant fluids. Fourteen experiments were performed with the trap method for the detection of small concentrations of enterovirus (Table 5). The reproducibility of the r values is not great, and this in turn influences the values of pobs. The value of Pobs does not, derive directly from r because the distribution of reeovered PFU among tubes may not exactly follow that predicted from the Poisson formula. Attempts to achieve better r values by removing the tral)s from the tubes with 0. ml of PMK cell suspension, rather than diluent, were unsuccessful. DISCUSSION The present communication reports part of a series of studies concerning the detection of viruses in foods. Because the minimal infectious dose in humans by the oral route is not known for enteroviruses, and because samples obtained from an outbreak of food-borne illness may have aged somewhat between the time that infection took place and the time of the tests, various methods of enhancing the sensitivity of tests for virus detection are being studied. Ultracentrifugation has been shown to be of value in detecting virus in sewage samples (Gravelle and Chin, 9) and stools (Peizer et al., 9), but reports have not indicated the quantities of virus which were detectable by the methods employed. Baron (957) quantitated the virus recovered with ultracentrifugation by various techniques, but did not determine how much virus was initially present. As a result, it has not been possible to predict the likelihood of detecting, by means of ultracentrifugation, enterovirus present at any particular titer in a very dilute suspension. An attempt has been made to generate some quantitative data regarding the efficiency of the preparative ultracentrifuge in concentrating laboratory suspensions of enterovirus at what have been designated arbitrarily as high, medium, and low titers. To make quantitation more precise, each virus suspension was passed through a membrane filter, usually at a porosity of less than twice the virus diameter, and was titrated immediately before ultracentrifugation. With both the number 30 and number 50 rotors of a Spinco model L ultracentrifuge, it has been found desirable to continue ultracentrifugation for a longer period of time than was predicted by the manufacturer's manual. During the course of these studies, there has been no indication that the two species of enteroviruses employed behaved any differently in the ultracentrifuge, and the results reported can probably be extrapolated to other members of the group. The enteroviruses are among the smallest agents known to be infectious for man, so it seems likely that the techniques described eould be applied to unknown virus suspensions with at least equal probabilityr of detecting other species (Gibbs and Cliver, in press). The evidence in favor of the use of traps in qualitative experiments is not unequivocal, but is taken to indicate that the trap method is at least as sensitive as ultracentrifugation without traps. Quantitative studies at low virus levels showed B3aron's method to be of no greater advantage than the trap technique, and in some instances the former was significantly less efficient. The trap technique is especially convenient for qualitative testing because the location of the pellet is indicated by the trap, and because the collection of the trap requires no mechanical action to dislodge it nor any more heating than is involved in momentarily holding the point in the tube where the trap is located against the palm of the hand. Sedimentation of large quantities of enterovirus into traps with very long ultracentrifugation times did not seem to result in any great degree of aggregation of the virus. Unknown samples of less than 0 ml can be

6 39 CLIVER AND YEATMAN APPL. MICROBIOL. concentrated in 0 mmi by means of the number 50 rotor. There is at least a 50% probability of detecting enterovirus present in the initial suspension at levels as low as 0. PFU per ml. Samples whose sizes exceed 0 ml may be divided among several tubes for the number 50 rotor, or may be doncentrated in the number 30 rotor. There is a 50% probability of detecting enterovirus by means of the number 30 rotor when the initial virus level is as low as 0.05 PFU per ml. ACKNOWLEDGMENTS This investigation was supported by Public Health Service research grant EF-000 from the Division of Environmental Engineering and Food Protection. The authors acknowledge with thanks the technical assistance of Mrs. Jimmie M. Martin. LITERATURE CITED BARON, S Ultracentrifuge concentration of poliovirus and effect of calf serum and gelatin. Proc. Soc. Exptl. Biol. Med. 95:70-7. BERG, G. 9. The food vehicle in virus transmission. Health Lab. Sci. :5-59. DARNELL, J. E., JR., R. Z. LOCKART, JR., AND T. K. SAWYER. 95. The effect of neutral red on plaque formation in two virus-cell systems. Virology :57-5. DIXON, W. J., AND F. J. MASSEY, JR Introduction to statistical analysis, p McGraw-Hill Book Co., Inc., New York. GODTFREDSEN, A., AND H. VON MAGNUS Routine diagnosis of enteroviruses using tissue culture techniques. Experiences in Denmark during Danish Med. Bull. :-9. GRAVELLE, C. R., AND T. D. Y. CHIN. 9. Enterovirus isolations from sewage: a comparison of three methods. J. Infect. Diseases 09: HEDSTRM, C. E., AND E. LYcEE. 9. An experimental study on oysters as virus carriers. Am. J. Hyg. 79:3-. HSIUNG, G. D The use of agar overlay cultures for the detection of new virus isolates. Virology 9: HSIUNG, G. D., AND J. L. MELNICK Plaque formation with poliomyelitis, Coxsackie, and orphan (ECHO) viruses in bottle cultures of monkey epithelial cells. Virology : KELLY, S., AND W. W. SANDERSON. 9. Comparison of various tissue cultures for the isolation of enteroviruses. Am. J. Public Health 5: MOSLEY, J. W., AND J. F. ENDERS. 9. A critique of the plaque assay technique in bottle cultures. Proc. Soc. Exptl. Biol. Med. 0:0-0. PEIZER, L. R., B. MANDEL, AND D. WEISSMAN. 9. An improved method for rapid laboratory diagnosis of poliomyelitis. Proc. Soc. Exptl. Biol. Med. 0: WALLIS, C., J. L. MELNICK, AND M. BIANCHI. 9. Factors influencing enterovirus and reovirus growth and plaque formation. Texas Rept. Biol. Med. 0: Downloaded from on March, 09 by guest