1976
]et.
TRAUTMAN, S. S. BREESE,JR.,B N D H. L. BACHRACH
Vol. 66
PREPARATIT’E ‘CLTRACESTRIFUCrBTIO,1’OF FOOT-ASD-MOUTH DISEASE VIRUS THROUGH IMRiPISCIRLE FLUID IXTERFACES INTO A CESIUM CHLORIDE DENSITY GRADIENT1 BY
RODEST R A U T & I 4 K ,
SYDNEY
s. BREESE,JR.,A N D HOWARD L. BACHRACH
Plum Island Animal Disease Laboratory, Agricultural Research Service, U . S . Department of Agriculture, Greenport, Long Island, ‘Yeto York Eeceived March 6,196.9
In order to effect purification in a short time, foot-and-mouth disease virus, type A, strains guinea pig GI3 and tissue culture 119, Mas centrifuged in a swinging bucket rotor from aqueous solutions through an immiscible organic layer into a short column of CsCl solution isodense with the virus. The organic layer was made of various combinations of volatile and nonvolatile solvents including trichlorotrifluoroethane, chloroform, diethyl phthalate, dibutyl phthalate, diethylhexyl sebacate, octanol, and butanol Selective penetration occurred when the organic separator was of loner density than the solvated virus particles and the protein contaminants of higher density were not too concentrated a t the upper interface. Removal of tissue particles of similar density and size to the virus could be achieved. The solvated density of the virus was increased by addition of CsCl and penetration was demonstrated by sedimentation or flotation for the density range 1.01-1.84 g./ml. Purification factors were computed from analytical ultracentrifuge patterns. Electron micrographs revealed hexagonal close packing of particles in phosphotungstic acid after two cycles of three hr. centrifugation at 37,000 r.p.m. in a 5-ml. SW 39 tube. Sodium ethylenediaminetetraacetate a t about 0.03 ‘11 partially stabilized purified virus and did not interfere with electron microscopy.
Introduction Organic fluids, both miscible and immiscible with water, have been used in various arrangements in preparative and analytical centrifugation. The miscible systems were selected to change the properties of the solvent, and the centrifugation was of the moving boundary t y ~ e . ~They , ~ will not be considered further; here the interest is with organic fluids that are immiscible with aqueous solutions of salts, proteins, and viruses. Such fluids have been applied for the batchwise purification of biological fluids by the selective denaturation of undesired proteins. Six other centrifugal uses of immiscible fluids, described in the literature, are shown schematically in Fig. 1 A-F, together with the one of this paper in G. In A, an organic fluid is used to prevent the collapse of the tube.4 In B, an organic phase below the aqueous solution avoided pelleting solute onto the lusteroid tube, enabling better resuspension.6 A similar application in the analytical cell (Fig IC) provided a second meniscus for sedimentation equilibrium measurements such as depicted in the schlieren diagram.6-* Figure 1D illustrat,es the proposal of Stenesh, et GZ.,~ for density measurements. A compression gradient in the organic phase caused the aqueous drops to move upward to a radius corresponding to their own density. Of direct relevance t o the present work is that of Bendet, et aZ.,iO (Fig. 1E) where the solute of the (1) Presented at t h e 3fitii National Colloid Symposium, Stanford University, June, 1962. (2) E. D. Rees and S J. Singer, a r c h . Bzochem. Bzophys., 63, 144 (1956). (3) H. L. Crespi, R. A. Uphaus, and J. J. Kate, J. Phys. Chem., 60, 1190 (195G). (4) L. Levintow and ,J. E. Darnell, Jr., J . Bzol. Chem., 236, 70 (1960). ( 5 ) D. R. Stanworth, K. James, and J. R. Squire, Anal. Bzochem., 2, 324 (1961). (6) N. K. Schachman. “Ultracentrifugation in Biochemistry,” Academic Press, New York, iY.Y., 1959, p. 186. (7) D . A. Yphantis, Ann. X. Y . Acad. Scz., 88, 586 (1960). ( 8 ) J. E. Hearst, J . B Ifft, and J. Vinograd, Proc. S a t l . Acad. Scz., r 8..47, 1015 (1961). (9) Described by Schachman, ref. 6 , p. 178 (10) I. J. Bendet, C. E. Smith, and M. 9. Lauffer, Arch. Bzochem. Bzophys., 88, 280 (ISSO).
upper aqueous phase penetrated the organic fluid. The backward displacement of the aqueous-organic interface was measured to calculate the solute hydrodynamic volume. The shape of the centrifuge cell magnified the displacement. The analytical ultracentrifuge was used and purified preparations were required. For Southern bean mosaic virus and E. coli, the density of the organic phase had to be less than the particle hydrodynamic density, corresponding to 0.98 ml./g. and 3.3 ml./g. hydration, respectively. “Differential flotation” of living cells was proposed by Ballentine and Burfordll (Fig. 1F). The organic phase density had to be between those of the cells to be separated. Kegligible solvent was carried down, permitting the sedimented cells to be freed from original growth medium. Criteria ciled by them for the organic phase were: (a) suitable density, (b) negligible solubility in mater, (c) physiological inertness, (d) low viscosity, and (e) low volatility. Diffusion pump oils such as phthalic acid esters and di-2-ethylhexyl sebacate were recommended. The arrangement described in this paper is shown in Fig. 1G,-here the solute (virus) must penetrate the organic phase and emerge into a CsCl solution adjusted to the isodensity of the solute. The organic separator permits the use of a column of CsCl small enough12 so that sedimentation occurs in a short time. Relatively large volumes of virus can be layered on top of the organic separator. Because the hydrodynamic density of protein contaminants is greater than that of the virus, the process is not principally differential flotation. Rather, organic fluids mere chosen to permit differential penetration. For the virus of interest, foot-and-mouth disease virus (FMDV) , the batchwise shaking of infectious aqueous fluids showed that chloroform,13 chloroform and butanol, or (11) R . Ballentine and D. D. Burford. Anal. Bzochem., 1, 26.3 (1960). (12) K. E. Van Holde and R . L. Baldain. J . P h y s . Chem., 62, 734 (1958). (13) G. Pyl, Arch. Exptl. VeteTinaefmed, 7, 238 (1953).
Oct., 1962
ULTRACESTRIFUGATIOS THROUGH
trichloro trifluoroethauie14 resulted in purification without loss of infectivity, while trichlorotrifluoroethane, with or without heptane, selectively separated virus from unassenbled protein subuiiits.16 Even though these chemicals are volatile, it mill be shown that they caii be employed. The development of isodensity centrifugation for the analytical study, as well as purification, of viruses vias initiated by Meselson, et al.,17and has been reriewed, along with moving zone density gradient methods, by Spinco.lg Studies on FMDV indicated that the CsCl isodensity value was 1.43 0.01 g./ml., which is significantly higher than that of most protein contaminaiits of density about 1.33 g:/m1.I9 The CsCl phase in Fig. 1G thus acts in a different la1 flotation manner allowing further purification and concentratioii without pellet formation. A virus is a useful colloid for studying the processes inr olved in penetrating interfaces because its infectivity assay permits measurement of concentration changes as great as 106-fold, even in the presence of other protein and nucleoprotein contaminant s. This paper presents data on such penetration by FLIDT‘ to derelop preparative procedures for its concentration and purification from crude harvest fluids. Experimental
IMIJIIBCIBLE FLUID
1977
B/?J
INTERFBCES
00
O O
C
A
*
Virus Source and Assay.-Laboratory stock FMDV, type A, &rain OB, highly adapted to guinea pigs, was inoculated into the hind pad3 of guinea pigs and both the infectious vesicular fluid (VF’) and foot pads (FP) were harvested 24 hr. later. Infectivity titrations were made in suckling micez0 using decimal dilutions with a litter of 10 mice per dilution and with litter mates randomized as suggested by SubakSharpe.2’ Nice were inoculated intraperitoneally with 0.03 ml. of the dilution. End-points, computed by the Spearman-Karber RIethod,zz had a standard deviation of 0.2 log unit. Some of the organic fluids killed suckling mice when injected undiluted, but none did so a t 10-1 dilution; hence there was no interference with the virus assay. CsCl solutions and normal serum also had no deleterious effect on the mice. Tissue culture ( T C ) adapted FhIDT, type -4,strain 119, was collerted from infected bovine kidney epithelial cell cultures, precipitated with methanol, resuspended, and ~1arified.l~This concentrated TC virus solution (SP,1,) was titrated by the plaque assay method23 in 4-oz. prescription bottles, using a 0.1 ml. inoculum. In this series of experiments there was a standard deviation of about 20%. SP,I, or VF purified further by organic solvent extractions are denoted as aqueous phase (AqPh).l4 Solutions.-Trishydroxymethylaminoethane buffer solutions were used a t 0.16 31, except for electron microscop . CsCl and disodium ethylenediaminetetraacetate ( E D T I ) were used as obtained from Fisher Scientific Co., Fairlavn, N. J. The pH of these solutions was adjusted to 7.4-7.8 before use. (14) H. L. Bachrach and S. S. Rreese, Jr., Proc. Soc. Exptl. Biol. X e d . , 97, 659 (1958). (1.5) M ,Mussgay, Z. H u g . Infeektionskrankh., 146, 48 (1959). (16) F. Brown and B. Cartwright, J. Inzmunol., 86, 309 (1960). (17) M. Meselson, I’. TV. Stahl, a n d J. Winograd, Proc. S a t l . A c a d . Sci., U. S., 43, 581 (1957). (18) “Spinco Technical Reviews. An introduction t o density gradient centrifugation,” Beckman Instruments, Inc., Spinco Division, Palo Alto, Calif., 1960. (19) R . T r a u t m a n and S. S. Breese, Jr., J. Gen. Microbial., 27, 237 (1962). (20) H. H . Skinner, TT. &I. Henderson, a n d J. B. Brooksby, ,Velure, 169, 794 (1952). (21) H. Subak-Sharpe, Arch. Ges. Virusforsch., 11, 1 (19G1). (22) See review by :D. J. Finney, “Statistical Method in Biological Assay,” Hafner Publ. Co., New York, N.Y . , 1952. (23) H. L. Bachrach, J. J. Callis. W. R. Hess, a n d R . E. P e t t y , Virology, 4 , 2!24 (1957).
“ 0
0
E
F
G
Fig. 1.--Arrangements for immiscible organic fluids and aqueous solutions in ultracentrifugation. In each grou the left hand figure 8how the initial loading; the right t a n d , after centrifugation; the purpose of the organic fluid (cross hatched) is to: A, occupy spaced; B, provide a liquid bottom;; C, provide a second meniscus6-8 (schlieren diagram a t extreme right); D, make a compression gradientg for aqueous droplets (solid circles); E, indicate hydrated volunielO; F, cause differential flotationll; and G, separate aqueous columns (this paper). Open circles represent faster component of higher density than crosses of slower s-rate and lower density. The organic chemicals were used without purification or water saturation. Trichlorotrifluoroethane (F) was obtained from Allied Chemical Corp., Baton Rouge, La., as “Genetron 113”; di-2-ethylhexyl sebacate (S) from Consolidated Vacuum Corp., Rochester, N . y., as “Octoil-S”; diethyl phthalate (E) and dibutyl phthalate (B) from Eastman Organic Chemicals, Rochester, N. Y.; chloroform (C); n-octanol (0),and n-butanol (A) from Fisher Scientific Co. The lusteroid tubes were softened by E in 1day, and by B in 2 weeks. The other chemicals had no effect. Density Measurements.--8queous solutions were measured in bromobenzene-m-xylene gradients a t 25‘ in 100-ml. graduated crlinders by the method of Jacobsen and Linder~trplm-Lang,2~ using standards determined by pycnometry. Organic solutions were measured in analogous manner in aqueous salt gradient columns. The “nominal” density a t 25“ of mixtures mas computed by linear interpolation by volume of handbook values for the density of the chemicals used and was found in agreement with the measured value. Because of vo1:itility and partial miscibility, the “nominal” density is used to denote the composition initially mixed. Preparative Ultracentrifugation,-,4 Spinco (Beckmnn Instruments, In(%.,Spinco Division, Palo Alto, Calif .) swinging bucket rotor, S W 39, was used in the Model L ultracentrifuge operated at 37,000 r.p.m. at 4-10” without brake. I n those cases in which tubes were to be centrifuged further, the rotor was slowly reaccelerated, taking about 6 min. to reach 37,000 r.p.m. The 5 ml. lusteroid centrifuge tubes were filled as follows (Fig. IG): first, 0.9 ml. of density 1.42 g./ml. CsCl solution (- 40yo w./m.); next, 0.9 ml. of the desired organic fluid mixture was layered; then, 3.3 ml. of the virus solution was slowly discharged onto the center of the organic meniscus. After centrifugation, the tubes were removed to a 4’ room and inspected for light scattering, using a narrow light beam. A tightly-banded light scattering zone (LS) a t a density of about 1.43 i 0.01 was indicative of FMDV.19 Fractions were removed by pipetting from the top with a Pasteur pipet, from a puncture in the side with 22 gage needle on a 1-ml. syringe, or by drop-out from a puncture in the bottom, depending upon which fractions were desired without contamination by the others. Most fractions were assayed for infectivity in addition t o noting the presence of LS and the location of debris and protein gel. ( 2 4 ) C. F. Jaoobsen and IC. Linderstr#m-Lang, Acta Physaol. Scand.. 1, 149 (1940).
R.TRAUTMAN, S. S. BIZEESE, JR.,AXD 13. L. BACHRACH
1978
Vol, 66
TABLE I EFFECTOF
lY0 (m./v.)
EDTA O N STORAQE OF PURIFIED FMDV UNDER VARIOUS CONDITIONS Entries" are drop in titer in log units Time of storage0 -1-4
7 -
------C-l
FMDV citrain
Conditions
None
day---
EDTA
-----0-1
wk.-------
None
EDTA
None
wk.-EDT-4
wk.
---4-16 None
--EDTA
Dialyzed 0.2(32) 0.3(18) 1.5'(7) 1.0r(16) 3.ac(7) 1.9'(4) In 3.4 111 CsCl 0.7(15) 0.1( 6) A119 Dialyzed 0,5'(5) 0.3'(9) 2.2(13) 0.9(14) 1.Sc(4) 1,8'(11) 4.3'(1) 2.3'(9) A119 In 3.4 iM CsCl 0.4( 1) 0.3( 5) 1.5( '2) 0.4( 2 ) 2 . 1 ( 3 ) 0 . 2 ( 4) >3.7(1) 0.6(1) A119 Dialyzed, stored -60" Number in parentheses is the number of values averaged. * Stored a t 4" except as noted in last row. No significant difference with or without CsC1, hence all values were averaged. A/GB
.4/GI3
Tubes with organic fluid densities above 1.2 g./ml. frequently were difficult to remove from the buckets. This was remedied in later experiments by using 0.2 ml. of HzO in the bucket, but sometimes tube creases resulted. Isodensity centrifugation without an organic fluid separator, for control or prior purification, was performed using a modification of the transient method of Matthews25 and of Trautman and Breese.19 In this, up to 3.8 ml. of the virus solution was layered over 1.5 ml. of a uniform CsCl solution of density 1.42 g./ml. After three hours centrifugation, the FMDV LS zone was below most of the debris, yet not pelleted. Analytical Ultracentrifugation.-A Spinco Model E ultracentrifuge equipped with phaseplate schlieren optics was used for moving boundary experiments to determine sedimentation rates and relative concentrations. Both 12 and 30 mm. double sector epon centerpieces were used. Photographs were measured on an optical comparator according to methods described elsewhere.28 Electron Microscopy .-Virus suspensions, dialyzed against 0.2 M NH40Ac with or without 1% (w./v.) EDTA4,were diluted to an awromiate level with 0.2 M (NHdLCOa for spraying or si&ading on formvar coated .copper mesh RcreeIis. Phosphotungstic acid (2y0)brought to pH 7.4 with 1 h KOH wab mived with an equal volume of t,he diluted virus before mounting on screens. Electron micrographs were taken at approximately 26,000X in an RCA EMU-3H electron microscope operated at 100 kv.
Results Controls.--Since FMDV is a riboiiucleic acid nucleoprotein, it was considered that Jig++ might afford some stabilization in purified preparations, as with riboso~nes.~~ I'relirninwy data indicated that there was no improvemerit M ith 0.02 AI Rig++. Hence, a chelating agent, 1% (w./v.) EDTA, was tried. The cumulative results for both virus strains that had at least one cycle of CsCl isodcnsity centrifugation are shown in Table I. Thc virus was dialyzed against a suitable buffer to remore CsCl as indicated. EDTA improved stability at 4" even for those samples remaining in CsC1. The TC A119 strain is seen to be less stable than A/GB, hence the 0-1 day storage values have been listed separately to show that the loss was not too severe for practical use. Over one week storage at 4' gave too great a loss, even though reduced with EDTA. At -60°, purified virus can be stored for long periods if EDTA is present. EDTA is now routinely used in all solutions, including the CsCl during the centrifugation. Because bottom fractions below an organic chemical were to be assayed for infectivity in this study, it was necessary to determine the contamination that could be expected without centrifuging. In each of two tubes, layered with infectious fluid ( 2 5 ) R. E. F. Matthcws, Nature, 184, 530 (1959). (26) R. Trautman, J . l'hys. Chem., 60, 1211 (1956). (27) M. L. Petermann. J . B d . Chsm., 236, 1998 (1960).
above a mixture of F and S organic liquids and left standing 66 hr. at 4") < l O - 3 % of the virus above the organic phase was found in the bottom CsCl phase. This is 1/1000 as much as the 1% level adopted here for evidence of penetration under the centrifugal field. Factors in Efficacy of Virus Penetration of the Interfaces.-Using the arrangement of Fig. lG, various mixtures of the organic fluids were surveyed for their effectiveness as a barrier for prott'\in contaminants while permitting the penetration of FMDV. The 183 experiments having the upper (starting virus) aqueous solution at 1.01 g./ml. density were grouped in the various categorics in Tables 11-V and scored as successes or failures. The effect of density and composition of organic fluid, location of debris, and source of virus mere analyzed. A. Effect of Density and Composition of Organic Phase.-Table I1 gives the resdts grouped in the TABLE I1 EFFECT O F DENSITY OFTHE ORGANIC F L U I D ON PENETRATION BY FMDV FROM AQUEOUSSOLUTION OF DENSITY1.01 G./ ML. L i t ries
_~__
are number of surcessfuP runs/total in each group. Organio fluid density range,* g./ml.
1 02-
1 041.061.081.101.04 1.06 1 08 1 10 1.12 >1.12 - __ - __ ______ S/S 62/64 35/41) 3/38 10/18 1/6 >lyc recovery of infectivity in CsCl solution below organic flud or presence of 1,s zone. * Cnlculated for 25" from composition of mixture uscd. I
I _
density classes indicated :it8 the top, rcg:irdless of the chemicals involved or the location of debris. In many experiments, fractions were taken on either side of the light scattering (LS) zone in the bottom CsCl solution. The infectivity measurements confirmedl9 the identification of such LS with banded virus. Hence 17 experiments that had no infectivity assay were included on the basis of presence or absence of LS. For the 166 main experiments, the criterion for penetration was that >1% virus infectivity was recovered in the entire bottom CsCl solution regardless of LS. In Table I1 it is seen that, proportionately, success declines above 1.10 g./ml. organic phase density and drops sharply above 1.12 g./ml. I n Table 111, 151 runs between 1.04 nnd 1.10 g./ml. were classified according to the organic mixture used. There seems to be no combination of chemicals which has a significantly low ratio of success, hence organic fluids may be selected for other properties than merely passing virus.
Oct., 1962
ULTRACENTRIFUGATION THROUGH IMMISCIBLE FLUID INTERFACES
TABLE I11 EFFECT OF COMPOSITION OF THE ORGANIC FLUID OF DENSITY 1.04-1.10 O./ML. SOLUTION OF DENSITY 1.01 G./ML.
ON
1979
P~NETRATION BY FMDV FROM AQUEOUS
Entries are number of successfuln runs/total in each category Density diluent-
B
Dense organic component
(Dibutyl phthalate, I .0416')
A
0
8
(Di-2-ethylhexyl sebacate, 0.9095')
(Octanol, 0.8228')
I? ( trifhlorotrifluoroethane, 1.5635') ... 32/38 2/2 C (chloroform, 1.4801') 1/ 2 23/25 30/31 FCd ... 20/26 1/1 E (diethyl phthalate, 1,1133") 4/4 6/7 2/3 ..* Kone 5/5 a > I % recovery of infectivity in C H Csolution ~ below organic fluid or presence of LS zone. d Used in equal proportions by volume. c d2b4 g./ml.
--.
(Butanol, 0.8057')
... ..
... 1/3
...
2/3
...
1/1
...
If the hydration of FMDV is 1 ml./g., similar to that measured by Bendet, et al.,'O for Southern bean mosaic virus, and if the 1.43 g./ml. isodensity in CsCl is assumed to be the dry density, then the hydrated density would be 1.18 g./ml. Hence it is concluded from Tables I1 and 111that, for penetration, the density of the organic fluid should be less than the hydrated density of the virus. This is consistent with the observations of Bendet, et uLIO Ruled out as factors for penetration are dry density and the nature of the organic fluids. Also eliminated are interfacial tension, viscosity, water solubility, and volatility, because of their wide variation in the mixtures of Table 111. B. Location of Debris after Centrifugation.-it is important for purification that any sediment of non-viral Contaminants be retained a t the upper interface. Table IV presents the results of 120 runs, using F, C, or FC as the dense component and O or S as the diluent where the location of debris was visible, Those having 0 as the. density diluent are listed in the first row, and those with S, in the second. Considering first the 0, it is seen that debris appeared a t the lower interface for all densities up through 1.11. Since this is the highest density that should be used to pass virus (from Table 11>, 0 is not a good diluent for F or C. But mixtures containing s, indicated in the second row, are seen to retain the debris a t the upper interface over 60% of the time. The lower portion of the table gives an analysis of the same S runs of the 1.04-1.11 density range in terms of volume, time, and density to determine the reasons for failure to retain debris a t the upper interface. It is seen that a higher percentage of successes was obtained the higher the density, the shorter the time, or the smaller the volume, oach factor being practically independent of the others. The mechanism by which the protein gel and debris got to the lower interface will be considered in the section on density measurements after centrifugation. C. Effect of the Nature of the Contaminants.-When impure preparations were studied, it was found that the minimum time required to detect a light scat,tering zone was increased over that with purified preparations, and the zone appeared higher in the CaC1 solution. This meant that the mere presence of a narrow light scattering zone of virus in the boltom layer after 3 hr. did not necessarily
SAL
...
...
Used in variable proportions.
TABLE IV EFFECTOF DILUENT,DENSITY, VOLUME,AND TIMEON THE RETENTION OF DEBRISAT UPPER INTERFACE Entries are the number of runs with visible debris or gel a t upper interface/total number of runs made --Organic Diluenta
0 (octanol) S (di-2-ethylhexyl sebacate) -Density
1.04-1.07
range-
1.07-1.11
fluid density range, b g./mI.1.02-1.04 1.04-1.11 > 1.11
0/4
0/31
5/5
1/1
46/74"
5/5
Vol.(ml.)
Time(hr.)
2-3.5 8/8 0.9-1.5 10/12 4-10 7/10 0.9-1.5 1/1 2-3.5 2/3 2 .O-4.0 0/1 2.0-4.0 4-10 1/10 17/29 F, C, or FC runs as dense component, either 0 or S a3 diluent. Calculated for 25" from composition of mixture used. Aqueous starting virus density was 1.01 gJxr.1. c These runs are further analyzed below. Q
mean that equilibrium had been reached. The virus could still be moving as a zone to its correct position. To demonstrate that the nature of the contaminants was involved, the same 151 runs between 1.04 and 1.10 density of Tables I1 and I11 were classified as to the magnitude of the recovery of infectivity and as to the source of the infectious fluid. The results are given in Table V. Consider first the left hand part of the table on guinea pig virus, strain A/GB. Of 13 runs in the first column on vesicular fluid that had been previously clarified by shaking with organic fluids (AqPh), 55% had a recovery of SO% recovery. Untreated vesicular fluid (VF) was best of the three crude starting fluids, with 38% of the 106 runs giving >SO% recovery. Equal or superior results were obtained with rnaterial that had undergone a t least one prior cycle of centrifugation (Purified). It appears that crude materials contain debris of higher s-rate than that of the virus which coats the upper interface impeding penetration of the virus, or there may be a variable denaturation of the debris. Since AqPh runs had lower recoveries than those with VF, the material that blocked the interface was not removed by organic extraction, but was increased by the partial denaturation of some of the protein. A check of this hypothesis was made by using fresh unclotted VF. No LS zone was seen after 3 hr. and only a faint one after 6 hr.
R. TRAUTMAN, S. S. BREESE,JR.,AKD H. L. BACHRACH
1980
TABLE V EFFECT OF THE SOURCE OF INFECTIOUS FLUID ON
THE
Vol. 66
RECOVERY OF VIRUS INFECTIVITY
-
Entries" are the number of runs made
Recovery range (%)
_AqPha_ _ ~ F PGuinea pig A/GB-- VFd
Purifiede
c
--Tissue SP,i,I
oulture AllD----AqPhg Purified8
>50 2 (1570)~ 1(9%) 40 (38%) 5 (42%) 0 0 2 1-50 4 (30%) 9 (82%) 55 (52%) 7 (58%) 2 2 1
