disease it was felt important to determine whether these viruses would also

PHYSICAL PROPERTIES OF TWO GROUP A COXSACKIE (HERPAN- GINA) VIRUSES WHEN PROPAGATED IN EGGS AND MICE AS DETERMINED BY ULTRACENTRIFUGATION AND ELECTRON MICROSCOPY ANGELA BRIEFS,1 SYDNEY S. BREESE, JR.,2 JOEL WARREN,2 AND ROBERT J. HUEBNER1 National Microbiological Institute, National Institutes of Health, Bethesda, Maryland and Department of Virus and Rickettsial Diseases, Army Medical Service Graduate School, Washington, D. C. Received for publication January 28, 1952 The following study was undertaken to determine certain physical properties of two types of Coxsackie group A (herpangina) viruses, which although antigenically different are closely similar in their clinical, epidemiologic, and pathologic manifestations (Huebner et al., 1951). Strain no. 93 (antigenically identical with Dalldorf Type 2) and strain no. 1816 (Huebner Type H3) were both recovered from cases of herpangina and can only be differentiated by serologic means (Beeman et al., 1952). In view of their many similarities as agents of disease it was felt important to determine whether these viruses would also possess similar physical properties. Furthermore, since one strain, no. 93, has been adapted to the chick embryo (Huebner et al., 1950) as well as to suckling mice, an opportunity existed for studying possible effects which propagation in two different hosts might induce in the physical properties of the same strain of virus. Finally, since the increasing number of reports on various groups of Coxsackie viruses contain little information regarding methods of isolation in a state of relative purity, a comparison was made of some of the common techniques of concentration and purification as applied to two types of viruses falling in the Coxsackie A group. MATERIALS AND METHODS Coxsackie virus, strain no. 93, was cultivated in both mouse tissue and amniotic fluid. It was used from the 8th to 10th mouse passage and from the 10th to 13th egg passage. Strain no. 1816 was grown only in suckling mice because thus far it has resisted attempts to adapt it to embryonated eggs. It was used from the 3rd to 5th mouse passage. Mouse muscle preparatios. Suspensions of mouse muscle were prepared as follows: Four day old mice were injected intraperitoneally with 0.04 ml of 103 suspension of infected mouse muscle. When the animals were moribund, within 4 days, the tissues were harvested. Approximately 300 g of muscle were used in each pool. Heads, tails, skin, and viscera were discarded and the remaining 1 National Microbiological Institute, National Institutes of Health, Public Health Service, Federal Security Agency, Bethesda, Maryland. 2 Department of Virus and Rickettsial Diseases, Army Medical Service Graduate School, Washington, D. C. 2.37

238 BRIEFS, BREibE, JR., WARREN, AND HUEBNER [VOL. 64 material (designated as "mouse muscle") was triturated to a 10 per cent suspension in physiologic saline with a Waring blendor. The blended suspension was kept in the refrigerator overnight. After one freezing and thawing, it was centrifuged at 3,000 rpm (1,300 avg g) for 15 minutes and the supernatant was again frozen and thawed twice. Following a second centrifugation at the same speed the resulting supernatant was twice extracted with ether. Each extraction consisted of 8 to 10 changes of ether in 20 to 40 per cent concentration. The ether was finally removed by vacuum, the water soluble phase frozen and thawed once, and then centrifuged at low speed for 15 minutes. The supernatant was spun in the Spinco Model L ultracentrifuge at 40,000 rpm (105,400 avg g) for 75 minutes, and the sedimented virus resuspended in 1/20th of the centrifuged volume in physiological saline. The suspension was kept in the cold overnight, frozen, thawed, and centrifuged at 10,000 rpm (6.590 avg g) for 10 minutes to remove large aggregates. In certain experiments, before being used for sedimentation studies in the analytical ultracentrifuge or for examination by the electron microscope, the supernatant was digested with 0.1 per cent twice crystallized trypsin for 1 to 2 hours at 37 C, filtered through paper (Fisher no. 9), and centrifuged again for 75 minutes at 40,000 rpm. This second high speed sediment was resuspended in physiologic saline, ph 7.0. In two experiments with mouse muscle protamine sulfate precipitation was used between the ether extraction and the first ultracentrifugation. Protamine sulfate was added to the ether extracted suspension to a final concentration of 5.0 mg per ml. The suspension was kept in the refrigerator for 30 minutes with frequent agitation and then centrifuged at 3,000 rpm for 15 minutes (Warren et al., 1949). The virus then was sedimented by high speed centrifugation as described previously. Normal mouse muscle was prepared in the same way from uninfected 7 day old mice. Amniotic fluid preparation. Following the 10th passage in eggs amniotic fluid was found to contain comparatively large amounts of no. 93 virus (Huebner et al., unpublished). The yield, nitrogen content, ph, infectivity, and complement fixation titer of amniotic fluid were compared in eggs of varying ages. Optimum virus content was obtained when the amniotic fluid was harvested from 12 day old embryos, which had been injected after 6 days' incubation at 37 C. The inocula consisted of a 20 per cent chick embryo suspension from the 10th to 13th egg passages. At harvest undiluted amniotic fluid was pooled from 450 to 500 eggs to make a lot of approximately 1,000 ml. The purification procedure then followed the same steps used for mouse muscle material. Amniotic fluid from 12 day old uninoculated eggs was used as control material since pilot experiments had shown that amniotic fluid from uninoculated eggs and eggs inoculated with normal chick embryo suspension had the same nitrogen content, ph, and water clear appearance. Measurement of virus content. Two methods were used to determine the quantity of virus in the preparations: infectivity titrations in suckling mice, and titration of antigen content by the complement fixation test. Infectivity titrations were made in tenfold dilutions, and a volume of 0.04 ml

1952] PHYSICAL PROPERTIES OF TWO COXSACKIE VIRUSES was injected intraperitoneally into groups of eight 4 day old mice. Within the critical dilutions 16 mice were used. All mice were observed for ten days after which the 50 per cent end point (LD60) was calculated by the method of Reed and Muench. Complement fixation tests were performed essentially in the manner described by Bengtson (1944), except that overnight icebox incubation was employed for primary fixation. Nitrogen determinations were conducted by the micro-kjeldahl method. Analytical centrifugation and electron microscopy. Sedimentation patterns were obtained both by refractive index and ultraviolet absorption (2500 A) methods using a Spinco Model E ultracentrifuge. For most experiments the suspensions were concentrated 500 to 600 times before analysis. Electron micrographs were made with an RCA model EMU electron microscope using a self-biased gun and an objective aperture. The specimens were shadowed with chromium at an angle of tan-' 1/5. For these studies amniotic fluid was usually concentrated about 70 times, mouse muscle about 40 times. Single particles were measured. RESULTS An initial comparison was made of different procedures for purification of the two strains of viruses. These comprised ether for removal of lipoids, protamine for the precipitation of nonviral protein components, and different cycles of high speed centrifugation. In certain experiments, trypsin digestion was employed between the first and the second cycle. Table 1 illustrates the findings of an experiment in which the same virus pool was purified by four different methods. The results of method A and B, table 1, were obtained with mouse muscle when ether extraction was used with or without protamine precipitation. There was no measurable loss of infectivity by both methods, but method B gave a slightly higher LD6o/g nitrogen. Other preparations, however, gave the same results for both methods. Amniotic fluid, which has considerably less lipoids and nonviral nitrogen in the crude state, gave similar infective and antigenic levels when purified by any of the three sequences shown in table 1 (methods A, B, D). Previous experience has shown that electronic visualization of particles smaller than 40 mu in tissue suspension is facilitated if nonviral debris is reduced in the preparations by enzymatic digestion (Warren et al., 1950). Virus obtained by methods A and B (table 1) was subsequently digested with crystalline trypsin in the manner described previously. The undigested resuspended virus concentrates obtained from either muscle or amniotic fluid were yellow and turbid. However, the filtrates following tryptic digestion were water clear and colorless. These methods which yielded concentrates of increased infective titer also gave satisfactory complement fixing antigens. The complement fixing titers refer to material concentrated 5 to 600 times as indicated in the tables. Comparison of the complement fixing titer with the infective titer does not show a consistent correlation between the two. However, in every case, there was a considerable increase in the complement fixing titer with increase in infective titer. In order to evaluate the two different hosts as virus sources of strain no. 93 239

240 BRIEFS, BREESE, JR., WARREN, AND HUEBNER [VOL. 64 and compare the effectiveness of the methods used upon these materials, it should be noted that crude mouse muscle was a 10 per cent suspension, whereas amniotic fluid was used undiluted. The infectivity per g nitrogen of the two crude materials averaged about 1.3 logs higher for mouse muscle than for amniotic fluid. By comparison the infectivity per g nimtrogen of the purified mouse material was about 2.0 logs higher than for purified amniotic fluid. Therefore, mouse material TABLE 1 Effects of the sequence of basic purification procedures on virus from mouse muscle tissue and amniotic fluid A B c D npre.i592t5t4 1) Extracted 1) Extracted STSTART2) wtith ther with ether 1) Centrifuged1)ecpt- Centhriue 2) Precipitated at 40,000 rpm IANG prowthmn a ooo p with protamine 2) Reupn-2) Centrifuged atio inuphnil. at 40,000 rpm 3) Extracted at 40,000 rpm oleia p 4) (uspend) with ether 3) Resuspend- (Lo roamn Reu en- 10 X conc 20 X conc 5 X conc 10 X conc (strain~ ~ ~~~~~~2 Cetrfue 1816)~ ~ ~~~~~~~~3 Complement atxresusende 3Tr 40/81/16 0 3 Cetrfue /3 ed/2 Mouse muscle Titration in mice 7.7 8.5 9.2 7.5 7.4 (strain no. (Log LDio)* 1816) - Complement fix- 1/8 1/16 1/32 1/8 1/2 ation titer* Nitrogen (mg/ml) 1.05.18.22.11.15 LD&o/g Nt (Log) 12.0 13.6 14.2 12.8 12.6 VlXeconc fsxconc Xconc Amniotic fluid Infective titer 5.8 6.5 6.4-6.4 (strain no. (Log LD.o) 93) Complement fix- 1/4 1/8 1/8-1/8 ation titer Nitrogen (mg/ml).51.16.16 -.22 LDjso/g N2(Log) 10.5 11.6 11.6-11.5 *Values for the concentration indicated. offered a better virus source and the methods used were about 5 times more effective on mouse material than on amniotic fluid. Sedimentation analysis. The strain or the source of virus used made no difference in its sedimentation constant. Both types of virus studied showed one relatively fast moving boundary of approximately 150 S (table 2). This boundary was consistently found in preparations of sufficiently high concentration to give a sedimentation pattern.

1952] PHYSICAL PROPERTIES OF TWO COXSACKIE VIRUSES 241 Sharpest and highest boundary peaks were obtained with mouse muscle which invariably contained more virus. For 6 determinations the average value for the fast moving components of strain no. 93 from mouse muscle was 148 S, while TABLE 2 Physical and chemical data on crude and purified suspensions of virus from mouse muscle and amniotic fluid COMPLEMENT SED. CONST. VIRAL STRAIN AND SOURCE MATERIAL (LOG LDbo)* (LOG) FIXATION TITER* (Sw X 1011 SEC) SZ S IZE INFECTIVITY LDSO/G NI (SII 11 PARTICLE Strain no. 93 10% susp 7.0 11.4 1/4 (mouse muscle) 600 X conc 9.4 14.3 1/128 150.4 35.6 40.8 18.6 10% susp 7.7 12.0 1/4 600 X conc 9.5 15.0 1/256 150.5 35.7 76.7 26.1 - Strain no. 93 100% susp 5.8 10.4 1/2 (amniotic fluid) 366 X conc 8.2 12.8 1/64 152.2 36.8 54.2-100% susp 6.2 10.9 1/4 600 X conc 7.3 12.3 1/128 145.8 36.8 Strain no. 1816 10% susp 7.5 11.9 1/8 (mouse muscle) 600 X conc 9.7 1/128 148.6 36.8 87.0 51.1 18.7 10% susp 7.7 12.1 1/8 320 X conc 10.0 14.7-143.1 77.4 36.8 54.2 18.7 10% susp 8.0 12.5 1/8 600 X conc 10.0 14.9 1/256 150.5 37.7 74.0 33.2 16.7 * Values for the concentration of material indicated. t Measured by electron microscopy. in two determinations the value for the same strain from amniotic fluid was 149 S. Strain no. 1816 from mouse muscle had an average of 148 S for 5 determinations. Most of the preparations also had one or two slower moving components.

242 BRIEFS, BREESE, JR., WARREN, AND HUEBNER [VOL. 64 Downloaded from http://jb.asm.org/ Figure. 1. Coxsackie no. 93 prepared from amniotic fluid. Chromium shadowed. Dow latex balls added. Figure 2. Coxsackie virus no. 93 prepared from amniotic fluid. Chromium shadowed Figure 3. Coxsackie virus no. 93 prepared from mouse muscle. Chromium shadowed Figure 4. Coxsackie virus no. 1816 prepared from mouse muscle. Chromium shadowed Figure 5. Particles from normal mouse muscle. Chromium shadowed Figure 6. Particles from normal amniotic fluid. Chromium shadowed The constants were not uniform but appeared to be grouped about 2 modes. In one of two runs with infected amniotic fluid and in 10 of 11 similar runs with infected muscle a component of 40 S average was found. The third component of 80 S average was observed only in infected mouse muscle (10 of 11 runs). The presence of these two boundaries was not related to the method of prepara- on December 9, 2018 by guest

19521 PHYSICAL PROPERTIES OF TWO COXSACKIE VIRUSES 2493 tion nor their variability to the final concentrations. The 40 S component usually did not seem to be affected by the concentration of trypsin and length of digestion which were used, whereas the component of 80 S in mouse muscle was diminished by digestion. In one experiment with strain no. 93 and one with strain no. 1816 mouse muscle, the comparative decrease after tryptic digestion was measured planimetrically; the component of 80 S was found to be decreased by about 50 per cent after digestion. Noninfected control preparations of mouse muscle and amniotic fluid were centrifuged in concentrations ranging from 300 to 800 times and had nitrogen contents from 0.084 to 0.174 mg per ml. It was impossible to detect any boundary by either adsorption or refraction in 7 runs. Observations by electron microscopy. No difference between the sizes of the infectious agent from two different strains nor between the same strain from different hosts could be found. Cont,entrates from the two virus strains from both host sources revealed particles which were nearly spherical and quite uniform in size ranging from 36 to 38 m,u. They are shown in figures 1, 2, 3, and 4. Amniotic fluid preparations gave clearer pictures than mouse muscle material. A second particle with a size of 18 my was seen in strains no. 93 and no. 1816 muscle preparations and in highly concentrated amniotic fluids infected with no. 93. This same size particle was also observed in normal mouse muscle and occasionally in normal amniotic fluid (figures 5 and 6). It would appear that the 37 mu particles corresponded to the 150 S boundary and that they were virus particles. They were found in all preparations of both strains in mouse muscle and in amniotic fluid infected with strain no. 93. They were not found in normal material. The 18 m,u particles were assumed to correspond to the 40 S boundary and were considered to be normal components. No particles could be definitely identified to correspond to the 80 S boundary. DISCUSSION The efforts reported in this manuscript should not be regarded as an attempt to characterize with respect to size the anomalousgroupsof virusesnow "lumped " under the term Coxsackie or C virus. Our main purpose was to obtain information concerning the size of two members of a single group of viruses which produce identical pathological responses in mice and which are responsible for a specific nosological clinical entity in man-herpangina. The known members of this group of viruses (H1, H2, H3, H4, Dalldorf Type 2, and High Point or Texas strains) differ in that they produce distinct immunological responses; but in all other respects they appear to represent a homogeneous group of viruses (Beeman et al., 1952). The sedimentation constant of approximately 150 S found for the infectious particle of both strains agrees well with the values given by Melnick et al. (1951) for members of the same group for equal times of centrifugation. The diameter of 37 m,u found for the infectious particle in electron micrographs may be used in conjunction with the sedimentation constant to calculate a particle density

244 BRIEFS, BREESE, JR., WARREN, AND HUEBNER [VOL. 64 of approximately 1.2 assu an unhydrated sphere (Svedberg and Pederson, 1940). The average diameter we have determined for the two viruses by measurement of single particles (36 to 38 my) is larger than has been reported for another member of the same group, namely the High Point (Texas I) strain. Melnick et al. (1951) assign a diameter of 28 m,u by ultracentrifugation and 15 to 23 m,u by ultrafiltration, and Himmelweit et al. (1950) give a value of 10 to 15 m,u by ultrafiltration. Using the same value for partial specific volume as Melnick et al. (1951), i.e. 0.77, our ultracentrifugal data would give a calculated diameter for the strain no. 93 and strain no. 1816 viruses of 30.2 m,u. The slower moving boundaries found in the sedimentation patterns of the infectious materials have not been completely identified with particles seen in the electron micrographs. The 18 m,u particles found in electron micrographs of mouse muscle material of both strains, in normal mouse muscle, and occasionally in normal amniotic fluid presumably could be responsible for the 40 S boundaries. Similar boundaries have been found in preparations of normal mouse brain (Weil et al., 1952), and in normal chick embryo (Kahler and Bryan, 1943). Boundaries with mobilities of approximately 80 S were found in normal and infected chick embryo suspensions (Taylor et al., 1942). Since the preparations of normal mouse muscle and normal amniotic fluid showed no sedimentation boundaries in repeated attempts, there was no exact way of confirming whether these two additional sedimentation boundaries were due to normal or pathological components. However, subsequent experiments in the separation cell showed that over 95 per cent of the infectivity sedimented with the 150 S boundary so that the two slower moving components are presumed not to be infectious (Breese and Briefs, unpublished). In purifying the two strains from mouse muscle and amniotic fluid, it was found that the use of protami'ne precipitation, in combination with ether extraction and repeated freezing and thawing, did not regularly increase the infectivity to nitrogen ratio. Furthermore, in two protamine preparations with the no. 93 strain, loss of virus titer was found. Therefore, although it had been previously employed for precipitation of similar viruses, protamine was omitted in subsequent experiments (Weil et al., 1952). Quigley (1949) mentioned the use of methanol precipitation in combination with ultracentrifugation for purification of other group A viruses but gave no estimate of the purity of his preparations. While our findings with respect to two members of the homogeneous group of viruses might possibly be extrapolated to other members of the same group, we do not believe they can be applied with any validity to other quite distinct viruses, such as the Connecticut 5 strain, which produces a different disease in suckling mice, pancreatitis in adult mice, and has been associated with epidemic pleurodynia in man (Shaw et al., 1950; Pappenheimer et al., 1950; Weller et al., 1950). The same can be said for other types of virus (Dalldorf Type 1, Powers, and Ohio strains) classified as C viruses because they behave somewhat similarly in suckling mice but which have not been identified satisfactorily with any specific illness.

19-021 PHYSICAL PROPERTIES OF TWO COXSACKIE VIRUSES 24j5 ACKNOWLEDGMENTS The authors wish to thank Dr. William C. Alford, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, for carrying out the nitrogen determinations, and Mr. Julius Kasel and Mr. Horace C. Turner, National Microbiological Institute, National Institutes of Health, for technical assistance. SUMMARY Two strains (no. 93 and no. 1816) of the Coxsackie group A viruses were purified from mouse muscle tissue and amniotic fluid (strain no. 93) with ether extraction, high speed centrifugation, and trypsin digestion. The concentration of the final material as determined by LDro/g nitrogen was in the average 2.5 logs higher, the purest preparations having an LD6o/g nitrogen of 10g. The size and sedimentation constant of the no. 93 strain in two host tissues were the same. For both strains the sedimentation constant of the component presumed to be the virus was in average 150 S. The particle size determined from electron micrographs was approximately 37 miu. Purified infected preparations contained several boundaries when examined in the ultracentrifuge, whereas control materials showed no sedimentation boundaries. However, particles smaller than the virus were found in micrographs of normal mouse muscle and probably represent one or more not infectious components, in quantity too small to give sedimentation constants. REFERENCES BEEMAN, E. A., HUEBNER, R. J., AND CoLE, R. M. 1952 Studies of Coxsackie viruses. Laboratory aspects of the group A viruses. Am. J. Hyg., 55, 83-107. BENGTSON, I. A. 1944 Complement fixation in rickettsial diseases. Technique of the test. Pub. Health Repts., 59, 402-405. BREESE, S. S., JR., AND BiuEFs, A. Unpublished data. HImmELWEIT, F., FINDLAY, G. M., AND HOWARD, E. M. 1950 The Size of Coxsackie viruses as estimated by filtration through gradocol membranes. Brit. J. Exptl. Path., 31, 809-812. HUEBNER, R. J., COLE, R. M., BEEMAN, E. A., BELL, J. A., AND PEERS, J. H. 1951 Herpangina-etiological studies of a specific infectious disease. J. Am. Med. Assoc., 145, 628-633. HUEBNER, R. J., RANSOM, S. E., AND BEEMAN, E. A. 1950 Studies of Coxsackie virus. Adaptation of a strain to chick embryos. Pub. Health Repts., 65, 803-806. HUEBNER, R. J., RANSOM, S. E., AND BRIEFS, A. Unpublished data. KAHLER, H., AND BRYAN, W. R. 1943 Ultracentrifugal studies of some complexes obtained from mouse milk mammary tumors and other tissues. J. Natl. Cancer Inst., 4, 37-45. MELNICK, J. L., RHIAN, M., WARREN, J., AND BREESE, S. S., JR. 1951 The size of Coxsackie viruses and Lansing poliomyelitis virus determined by sedimentation and ultrafiltration. J. Tmmunol., 67, 151-162. PAPPENHEIMER, A. M., DANIELS, J. B., CHEEVER, F. S., AND WELLER, T. H. 1950 Lesions caused in suckling mice by certain viruses isolated from cases of so called non-paralytic poliomyelitis and of pleurodynia. J. Exptl. Med., 92 169-190. QUIGLEY, J. J. 1949 Ultrafiltration and ultracentrifugation studies of Coxsackie virus. Proc. Soc. Exptl. Med., 72, 434-435.

246 BRIEFS, BREESE, JR., WARREN, AND HUEBNER [VOL. 64 SHAw, E. W., MELNICK, J. L., AND CURNEN, E. C. 1950 Infection of laboratory workers with Coxsackie viruses. Ann. Internal Med., 33, 32-40. SVEDBE:RG, T., AND PEDERSON, K. 0. 1940 The Ultracentrifuge. Oxford, The Clarendon Press, p. 395. TAYLOR, A. R., SHARP, D. G., BEARD, D., AND BEARD, J. W. 1942 Isolation and properties of a macromolecular component of normal chick embryo tissue. J. Infectious Diseases, 71, 115-127. WARREN, J., WEIL, M. L., Russ, S. B., AND JEFFRIEs, H. 1949 Purification of certain viruses by use of protamine sulfate. Proc. Soc. Exptl. Biol. Med., 72, 662-664. WARRN, J., WEIL, M. L., Russ, S. B., AND JEFFRIES, H. 1950 Applications of protamine precipitation in purification of certain viruses. Federation Proc., 9, 394 (abstract). WEIL, M. L., WARREN, J., BRE5ESE, S. S., JR., RUSS, S. B., AND JEFFRIES, H. 1952 Separation of encephalomyocarditis virus from tissue components by means of protamine precipitation and ensymic digestion. J. Bact., 63, 99-105. WELLER, T. H., ENDERS, J. F., BUCKNGHAM, M., AND FINN, J. J., JR. 1950 The etiology of epidemic pleurodynia: A study of two viruses isolated from a typical outbreak. J. Imimunol., 65, 337-346. Downloaded from http://jb.asm.org/ on December 9, 2018 by guest