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Relation of sialidase of influenza a viruses to viral particles as determined by electron microscopy
VIROLOGY
13, 1%18 (1961)
Relation
of Sialidase of Influenza A Viruses to Viral Determined by Electron Microscopy’
Particles
as
J. T. SET0,2 Y. NISHI,3 BETTY JANE HICKEY, AND A. F. RASMUSSEN, JR. Division
of Virology,
Department of Infectious Diseases, School of California, Los Angeles University Accepted
August
of Medicine,
17, 1960
We have measured the sialidase activity per virus particle of the following strains of influenza A : A/Swine/32, A/PR8/33, AI/FM1/47, A2/Japan305/57, and A2/Taiwan/ 58. Strains differed bv as much as a hundredfold in activity of individual particles. Japan 305 was the most active. INTRODUCTION
tine, 1957)) and the morphologic inhomogeneity of allantoic fluid preparations of A2 strains raised questions as to the validity of this assumption. In order to confirm the relationship of the sialidase found in chorioallantoic (CA) fluids of eggs infected with influenza viruses to the virus particle, particle counts were determined by direct electron microscopy and related to sialidase activity. The results, described herein, show that the unique sialidase activity of A2 strains is a property of the virus particle.
In a comparative study of the sialidase activity of representative strains of influenza A, we reported (1) that the A2 strains had greater sialidase activity than other A strains, and (2) that the sialidase of A2 strains and of swine was more stable to heat than was the hemagglutinin in contrast to the classic reaction of influenza viruses to heat in which the sialidase is more heat labile than is the hemagglutinin (Seto et al., 1959a,b). For these comparative studies, sialidase activities were related to hemagglutinin titers. Because the sialidase was closely associated with the hemagglutinin it was assumed that the sialidase was an integral part of the virus particle, as is so for the sialidases of influenza A, Al, and B strains previously studied (Hoyle, 1952; Davenport et al., 1959). However, the marked differences between the sialidases of A2 and other strains, the limitations of hemagglutination as an estimate of numbers of viral particles (Tyrrell and Valen-
MATERIALS
AND
METHODS
Virus Preparations The following strains of virus, A2/Japan 305/57, A2/Taiwan/58, A/PR8/33, A/ Swine/32 and Al/FM1/47, were prepared as described previously (Seto et al., 1959a, b). For this study the virus concentrates were purified by two additional cycles of differential centrifugation in a 40 Spinco rotor for 60 minutes at 25,000 rpm and for 15 minutes at 2000 rpm in an International model V centrifuge. The final pellets from 500-600 ml of infected CA fluid were resuspended in 4-5 ml of phosphate-buffered saline (PBS), pH 7.0, 0.01 M and dialyzed over night in the cold room against 1% ammonium acetate solution (pH 7). One preparation of Japan 305/;7 was further puri-
’ Supported by grants from Institute of Allergic a id Infectious Diseases and National Heart Institute, NIH, United States Public Health Service. ’ Present address: Department of Biology, LOS Angeles State College. 3 Permanent address : Department of Virology and Biophysics, Research Institute for Microbial Diseases, Osaka University, Japan. 13
14
SETO,
NISHI,
HICKEY
fied, prior to dialysis, by adsorption and elution from chicken red cells as follows: To 10 ml of a virus suspension, which was partially purified by differential centrifugation, 10 ml of 30% chilled chicken red cells was added at 4”. After allowing 2 hours at 4” for adsorption and for the red cells to settle, the supernatant was decanted; the remaining cell suspension was centrifuged and the supernatant was removed. The supernatants were pooled and subjected to a second adsorption with 5 ml of 30% chicken red cells as described. The final supernatant was removed and discarded and 5 volumes of PBS containing 100 units of penicillin per milliliter and 100 pg streptomycin per milliliter
AND
RASMUSSEN
JR.
was added to the packed cells from both the first and second cycles of adsorption. Virus was eluted from the cell suspension by incubating at 37” and periodically agitating the cell suspension for 11/2 hours. The cells were removed by centrifugation and the eluted virus was recovered in the supernatants. The virus suspension was centrifuged at 30,000 rpm for 1 hour and the pellet was resuspended in 2 ml PBS. Direct particle counts were made by the method of Williams and Backus (1949) with samples composed of the following: 20 parts of a standard polystyrene latex suspension
FIG. 1. Electron micrograph of PR8 influenza virus. Spray drop shadowed Polystyrene latex reference particles with a diameter of 260 rnw.
with
uranium
SIALIDASE
ACTIVITY
OF INFLUENZA
containing 5.0 X loll particles per milliliter (no. 586 supplied by Dow Chemical Company, Midland, Michigan), 25 parts of an 8% ammonium acetate solution, 10 parts of 0.1% bovine plasma albumin, and 45 parts of purified -virus. This preparation was sprayed on specimen grids with an H. E. Curry commercial nebulizer, shadowed with uranium, and examined with an Hitachi model HU-9 electron microscope. Sialidase Analysis Sialidase activities were determined as described previously (Seto et al., 1959a,b) and expressed as percentage of sialic acid
A VIRUSES
15
hydrolyzed by the determined number of virus particles per reaction flask. RESULTS
Electron
Microscopy
Electron micrographs of PRS, FMl, and Swine concentrates revealed that a high degree of purification was achieved by differential centrifugation and dialysis against ammonium acetate. The FM1 preparation required, in addition, homogenization in a Virtis homogenizer to disperse the aggregates of virus particles. A representative micrograph is shown of PRS (Fig. 1). On the other hand, preparations of the A2
FIG. 2. Electron micrograph of Japan 305/57 influenza owed with uranium. Polystyrene latex reference particles
virus (sample 1). Spray drop shadwith a diameter of 260 m/L.
TABLE
1
RELATION OF VIRAL PARTICLES AS MEASURED BY DIRECT COUNTS TO HB TITERS HA titera of concentrate
Virus
Dilution of concentrate for counts ~.__________
Particles counted per sample
Particles per ml
Ratio of particles H.4 titer
Japan 305
1.6 X lo5
20
181
4.0 x 10’2
2.5 X 10’
Japan 305
1.0 x 104
10 20
1125 405
2.0 x 10’2 2.0 x 10’2
2.0 x 108 2.0 x 108
Japan 305b
2.6 x 10”
5c 10
823 446
8.2 x 10” 7.7 x 10”
3.2 X 108 3.0 x 108
Taiwan
1.6 X lo5
20 40
167gd 1561
7.6 X 1Ol2 8.6 x lo’*
4.8 x 107 5.4 x 10’
PR8
4.1 x 104
20
440
6.3 x 10’2
1.5 x 108
PR8
8.2 X lo4
100 200
1730 683
1.8 X 10’3 1.7 x 10’3
2.2 x 108 2.1 X lo8
Swine
2.1 x 104
20 40
1363 686
5.0 x 10’2 5.0 x 10’2
2.4 X lo* 2.4 X lo*
FM1
5.1 x 103
10
1246
2.0 x 10’2
4 x 108
a Reciprocal of dilution. b Purified by adsorption onto and elution from red cells. c Formed aggregates. d Too many particles per droplet, for accurate counting.
60
VIRUSES
50
0 JAPAN
305/57
0 JAPAN
305/57
iI TAIWAN X FMI I
40
SWINE
0 PR8
30
20
IO
0 7
9 LOG,,
OF
IO
PARTICLES
FIG. 3. Sialidase
PER
12
II
REACTION
FLASK
activities of fire strains of influenza A expressed as percentage of sialic acid per virus particle. Open circles represent results with nonadsorbed preparation of Japan 305/57 containing small, 25-35 mp particles. Filled circles represent results with preparation of Japan 305/57 from which small particles have been removed by adsorption and elution. 16
to
SIALIDASE
ACTIVITY
OF INFLUENZA
strains contained debris and particles smaller (25-35 rnpu) than the elementary bodies (100-130 nip) (Fig. 2). Except for the occurrence of the small particles the strains were similar morphologically. Results of direct particle counts of representative preparations of the five strains are given in Table 1 together with their HA titers, dilutions of concentrates, numbers of particles counted per sample, and ratio of particles to HA titers. The reliability of the
A VIRUSES
17
counting technique can be seen from the counts of two dilutions of Japan 305, PR8, and Swine. With the exception of preparation no. 1 of Japan 305 and Taiwan, there is a rough relationship between HA titers and particle counts. The fivefold dilution of Japan 305, purified by adsorption onto and elution from red cells showed occasional aggregates, and the preparation of Taiwan diluted twentyfold contained so many par-
FIG. 4. Electron micrograph of Japan 305/57 influenza virus (sample l), adsorbed onto and eluted from red cells. Spray drop shadowed with uranium. Polystyrene latex reference particles with a diameter of 260 mp.
18
SETO,
titles per droplet count accurately.
NISHI,
HICKEY
that it was difficult
to
Xialidase Analysis Enzyme activities were determined immediately after dialysis of the virus concentrates before storing the samples in the cold room. Enzyme determinations of the five strains, Japan 305, Taiwan, PR8, Swine, and FMl, presented in Fig. 3, express activities as percentages of sialic acid hydrolyzed by a given number of virus particles per 4 ml substrate. It is evident that the A2 strains possess the greatest sialidase activity, next Swine and FM1 with the same activity, and finally PR8 with the least activity. Approximately 100 times the number of particles of PR8 were required to produce the level of activity shown by the A2 strains. Over an approximate tenfold range of particle concentration, enzyme activity was proportional to the log of the number of particles. Moreover, the slopes of the curves were similar with the exception of the curve represented by Taiwan strain (Fig. 3). The leveling off of the curves in the range of 5060% activity is interpreted as due to lack of an excess of substrate. To determine whether or not the small, 25-35 rnp, particles in A2 preparations possessed enzyme activity, an aliquot of a preparation of Japan 305 was further purified by adsorption onto and elution from chicken red cells. An electron micrograph (Fig. 4) shows that further purification removed the small particles from the virus particles. That the small particles did not contribute t,o the sialidase activity of A2 strains can be seen in Fig. 3. The curves of the nonadsorbed preparation containing small particles and the adsorbed and eluted preparations of Japan 305 are essentially similar. DISCUSSION
Morphologically, the A2 strains were similar to other A strains and were not filamentous as shown by Kilbourne (1959) for low egg passage A2 strains. Preparations of the A2 strains did contain debris and small particles, but these components did not contribute detectable sialidase activity. The similarity of the curves of the adsorbed and nonadsorbed preparations of Japan 305 also
AND
RASMUSSEN
JR.
shows that there were few if any particles like those described by Frommhagen and Knight (1959; Frommhagen et al., 1959)) which approached the size of influenza virus but did not hemagglutinate. The consistantly high sialidase activity, which is characteristic of A2 strains as compared to such A strains as FMl, PR8, and Swine, confirms our original observation (Seto et al., 1959a,b). The greater sialidase activity of A2 strains may be correlated either with direct particle counts or with HA titers. Whether the differences in sialidase activity are due to a qualitative difference in the sialidase moiety of the virus or to a quantitative difference in the number of sialidase moieties in each viral particle was not determined. ItEFERENCES DAYEXPORT, F. M., ROTT, R., and SCHAFER, W. (1959). Physical and biological properties of influenza virus components obtained after ether treatment. Fedex&ion Proc. 18, 563. FROMMHAGEN, L. H., and KNIGHT, C. A. (1959). Column purification of influenza v,irus. ViroEogy 8, 198-208. FROMMHAGEX, L. H., KNIGHT, C. A., and FREEXAS, N. K. (1959). The ribonucleic acid, lipid, and polysaccharide constituents of influenza virus preparations. Virology 8, 176-197. HOYLE, L. (1952). Structure of the influenza virus. The relation between biological activity and chemical structure of virus fractions. J. Hyg. 50, 229-245. KILBOURNE, E. D. (1959). Studies on influenza in the pandemic of 1957-1958. III. Isolation of influenza A (ilsian strain) viruses from influenza patients with pulmonary complica.tions. Details of virus isolation and characterization of isolates, with quantitative comparison of isolation methods. J. Clin. Invest. 38, 266-274. SETO, J. T., HICKEY, B. J., and RASXJSSEN, A. F., JR. (1959a). Some biological characteristics of Asian influenza isolates. Proc. Sot. Exptl. Biol. Med. 100,672-676. SETO, J. T., HICKEY, B. J., and RASMUSSEN, A. F., JR. (195913). Sialidase activity and related properties of influenza A2 viruses. Virology 9, 59% 611. TYRRELL, D. A. J., and VALENTINE, R. C. (1957). The assay of influenza virus particles by hemagglutination and electron microscopy. J. Gen. Microbial. 16,668-675. WILLIAMS, R. C., and BACKUS, R. C. (1949). Macromolecular weight determined by direct particle counting. J. Am. Chem. Sot. 71,4052-4057.
13, 1%18 (1961)
Relation
of Sialidase of Influenza A Viruses to Viral Determined by Electron Microscopy’
Particles
as
J. T. SET0,2 Y. NISHI,3 BETTY JANE HICKEY, AND A. F. RASMUSSEN, JR. Division
of Virology,
Department of Infectious Diseases, School of California, Los Angeles University Accepted
August
of Medicine,
17, 1960
We have measured the sialidase activity per virus particle of the following strains of influenza A : A/Swine/32, A/PR8/33, AI/FM1/47, A2/Japan305/57, and A2/Taiwan/ 58. Strains differed bv as much as a hundredfold in activity of individual particles. Japan 305 was the most active. INTRODUCTION
tine, 1957)) and the morphologic inhomogeneity of allantoic fluid preparations of A2 strains raised questions as to the validity of this assumption. In order to confirm the relationship of the sialidase found in chorioallantoic (CA) fluids of eggs infected with influenza viruses to the virus particle, particle counts were determined by direct electron microscopy and related to sialidase activity. The results, described herein, show that the unique sialidase activity of A2 strains is a property of the virus particle.
In a comparative study of the sialidase activity of representative strains of influenza A, we reported (1) that the A2 strains had greater sialidase activity than other A strains, and (2) that the sialidase of A2 strains and of swine was more stable to heat than was the hemagglutinin in contrast to the classic reaction of influenza viruses to heat in which the sialidase is more heat labile than is the hemagglutinin (Seto et al., 1959a,b). For these comparative studies, sialidase activities were related to hemagglutinin titers. Because the sialidase was closely associated with the hemagglutinin it was assumed that the sialidase was an integral part of the virus particle, as is so for the sialidases of influenza A, Al, and B strains previously studied (Hoyle, 1952; Davenport et al., 1959). However, the marked differences between the sialidases of A2 and other strains, the limitations of hemagglutination as an estimate of numbers of viral particles (Tyrrell and Valen-
MATERIALS
AND
METHODS
Virus Preparations The following strains of virus, A2/Japan 305/57, A2/Taiwan/58, A/PR8/33, A/ Swine/32 and Al/FM1/47, were prepared as described previously (Seto et al., 1959a, b). For this study the virus concentrates were purified by two additional cycles of differential centrifugation in a 40 Spinco rotor for 60 minutes at 25,000 rpm and for 15 minutes at 2000 rpm in an International model V centrifuge. The final pellets from 500-600 ml of infected CA fluid were resuspended in 4-5 ml of phosphate-buffered saline (PBS), pH 7.0, 0.01 M and dialyzed over night in the cold room against 1% ammonium acetate solution (pH 7). One preparation of Japan 305/;7 was further puri-
’ Supported by grants from Institute of Allergic a id Infectious Diseases and National Heart Institute, NIH, United States Public Health Service. ’ Present address: Department of Biology, LOS Angeles State College. 3 Permanent address : Department of Virology and Biophysics, Research Institute for Microbial Diseases, Osaka University, Japan. 13
14
SETO,
NISHI,
HICKEY
fied, prior to dialysis, by adsorption and elution from chicken red cells as follows: To 10 ml of a virus suspension, which was partially purified by differential centrifugation, 10 ml of 30% chilled chicken red cells was added at 4”. After allowing 2 hours at 4” for adsorption and for the red cells to settle, the supernatant was decanted; the remaining cell suspension was centrifuged and the supernatant was removed. The supernatants were pooled and subjected to a second adsorption with 5 ml of 30% chicken red cells as described. The final supernatant was removed and discarded and 5 volumes of PBS containing 100 units of penicillin per milliliter and 100 pg streptomycin per milliliter
AND
RASMUSSEN
JR.
was added to the packed cells from both the first and second cycles of adsorption. Virus was eluted from the cell suspension by incubating at 37” and periodically agitating the cell suspension for 11/2 hours. The cells were removed by centrifugation and the eluted virus was recovered in the supernatants. The virus suspension was centrifuged at 30,000 rpm for 1 hour and the pellet was resuspended in 2 ml PBS. Direct particle counts were made by the method of Williams and Backus (1949) with samples composed of the following: 20 parts of a standard polystyrene latex suspension
FIG. 1. Electron micrograph of PR8 influenza virus. Spray drop shadowed Polystyrene latex reference particles with a diameter of 260 rnw.
with
uranium
SIALIDASE
ACTIVITY
OF INFLUENZA
containing 5.0 X loll particles per milliliter (no. 586 supplied by Dow Chemical Company, Midland, Michigan), 25 parts of an 8% ammonium acetate solution, 10 parts of 0.1% bovine plasma albumin, and 45 parts of purified -virus. This preparation was sprayed on specimen grids with an H. E. Curry commercial nebulizer, shadowed with uranium, and examined with an Hitachi model HU-9 electron microscope. Sialidase Analysis Sialidase activities were determined as described previously (Seto et al., 1959a,b) and expressed as percentage of sialic acid
A VIRUSES
15
hydrolyzed by the determined number of virus particles per reaction flask. RESULTS
Electron
Microscopy
Electron micrographs of PRS, FMl, and Swine concentrates revealed that a high degree of purification was achieved by differential centrifugation and dialysis against ammonium acetate. The FM1 preparation required, in addition, homogenization in a Virtis homogenizer to disperse the aggregates of virus particles. A representative micrograph is shown of PRS (Fig. 1). On the other hand, preparations of the A2
FIG. 2. Electron micrograph of Japan 305/57 influenza owed with uranium. Polystyrene latex reference particles
virus (sample 1). Spray drop shadwith a diameter of 260 m/L.
TABLE
1
RELATION OF VIRAL PARTICLES AS MEASURED BY DIRECT COUNTS TO HB TITERS HA titera of concentrate
Virus
Dilution of concentrate for counts ~.__________
Particles counted per sample
Particles per ml
Ratio of particles H.4 titer
Japan 305
1.6 X lo5
20
181
4.0 x 10’2
2.5 X 10’
Japan 305
1.0 x 104
10 20
1125 405
2.0 x 10’2 2.0 x 10’2
2.0 x 108 2.0 x 108
Japan 305b
2.6 x 10”
5c 10
823 446
8.2 x 10” 7.7 x 10”
3.2 X 108 3.0 x 108
Taiwan
1.6 X lo5
20 40
167gd 1561
7.6 X 1Ol2 8.6 x lo’*
4.8 x 107 5.4 x 10’
PR8
4.1 x 104
20
440
6.3 x 10’2
1.5 x 108
PR8
8.2 X lo4
100 200
1730 683
1.8 X 10’3 1.7 x 10’3
2.2 x 108 2.1 X lo8
Swine
2.1 x 104
20 40
1363 686
5.0 x 10’2 5.0 x 10’2
2.4 X lo* 2.4 X lo*
FM1
5.1 x 103
10
1246
2.0 x 10’2
4 x 108
a Reciprocal of dilution. b Purified by adsorption onto and elution from red cells. c Formed aggregates. d Too many particles per droplet, for accurate counting.
60
VIRUSES
50
0 JAPAN
305/57
0 JAPAN
305/57
iI TAIWAN X FMI I
40
SWINE
0 PR8
30
20
IO
0 7
9 LOG,,
OF
IO
PARTICLES
FIG. 3. Sialidase
PER
12
II
REACTION
FLASK
activities of fire strains of influenza A expressed as percentage of sialic acid per virus particle. Open circles represent results with nonadsorbed preparation of Japan 305/57 containing small, 25-35 mp particles. Filled circles represent results with preparation of Japan 305/57 from which small particles have been removed by adsorption and elution. 16
to
SIALIDASE
ACTIVITY
OF INFLUENZA
strains contained debris and particles smaller (25-35 rnpu) than the elementary bodies (100-130 nip) (Fig. 2). Except for the occurrence of the small particles the strains were similar morphologically. Results of direct particle counts of representative preparations of the five strains are given in Table 1 together with their HA titers, dilutions of concentrates, numbers of particles counted per sample, and ratio of particles to HA titers. The reliability of the
A VIRUSES
17
counting technique can be seen from the counts of two dilutions of Japan 305, PR8, and Swine. With the exception of preparation no. 1 of Japan 305 and Taiwan, there is a rough relationship between HA titers and particle counts. The fivefold dilution of Japan 305, purified by adsorption onto and elution from red cells showed occasional aggregates, and the preparation of Taiwan diluted twentyfold contained so many par-
FIG. 4. Electron micrograph of Japan 305/57 influenza virus (sample l), adsorbed onto and eluted from red cells. Spray drop shadowed with uranium. Polystyrene latex reference particles with a diameter of 260 mp.
18
SETO,
titles per droplet count accurately.
NISHI,
HICKEY
that it was difficult
to
Xialidase Analysis Enzyme activities were determined immediately after dialysis of the virus concentrates before storing the samples in the cold room. Enzyme determinations of the five strains, Japan 305, Taiwan, PR8, Swine, and FMl, presented in Fig. 3, express activities as percentages of sialic acid hydrolyzed by a given number of virus particles per 4 ml substrate. It is evident that the A2 strains possess the greatest sialidase activity, next Swine and FM1 with the same activity, and finally PR8 with the least activity. Approximately 100 times the number of particles of PR8 were required to produce the level of activity shown by the A2 strains. Over an approximate tenfold range of particle concentration, enzyme activity was proportional to the log of the number of particles. Moreover, the slopes of the curves were similar with the exception of the curve represented by Taiwan strain (Fig. 3). The leveling off of the curves in the range of 5060% activity is interpreted as due to lack of an excess of substrate. To determine whether or not the small, 25-35 rnp, particles in A2 preparations possessed enzyme activity, an aliquot of a preparation of Japan 305 was further purified by adsorption onto and elution from chicken red cells. An electron micrograph (Fig. 4) shows that further purification removed the small particles from the virus particles. That the small particles did not contribute t,o the sialidase activity of A2 strains can be seen in Fig. 3. The curves of the nonadsorbed preparation containing small particles and the adsorbed and eluted preparations of Japan 305 are essentially similar. DISCUSSION
Morphologically, the A2 strains were similar to other A strains and were not filamentous as shown by Kilbourne (1959) for low egg passage A2 strains. Preparations of the A2 strains did contain debris and small particles, but these components did not contribute detectable sialidase activity. The similarity of the curves of the adsorbed and nonadsorbed preparations of Japan 305 also
AND
RASMUSSEN
JR.
shows that there were few if any particles like those described by Frommhagen and Knight (1959; Frommhagen et al., 1959)) which approached the size of influenza virus but did not hemagglutinate. The consistantly high sialidase activity, which is characteristic of A2 strains as compared to such A strains as FMl, PR8, and Swine, confirms our original observation (Seto et al., 1959a,b). The greater sialidase activity of A2 strains may be correlated either with direct particle counts or with HA titers. Whether the differences in sialidase activity are due to a qualitative difference in the sialidase moiety of the virus or to a quantitative difference in the number of sialidase moieties in each viral particle was not determined. ItEFERENCES DAYEXPORT, F. M., ROTT, R., and SCHAFER, W. (1959). Physical and biological properties of influenza virus components obtained after ether treatment. Fedex&ion Proc. 18, 563. FROMMHAGEN, L. H., and KNIGHT, C. A. (1959). Column purification of influenza v,irus. ViroEogy 8, 198-208. FROMMHAGEX, L. H., KNIGHT, C. A., and FREEXAS, N. K. (1959). The ribonucleic acid, lipid, and polysaccharide constituents of influenza virus preparations. Virology 8, 176-197. HOYLE, L. (1952). Structure of the influenza virus. The relation between biological activity and chemical structure of virus fractions. J. Hyg. 50, 229-245. KILBOURNE, E. D. (1959). Studies on influenza in the pandemic of 1957-1958. III. Isolation of influenza A (ilsian strain) viruses from influenza patients with pulmonary complica.tions. Details of virus isolation and characterization of isolates, with quantitative comparison of isolation methods. J. Clin. Invest. 38, 266-274. SETO, J. T., HICKEY, B. J., and RASXJSSEN, A. F., JR. (1959a). Some biological characteristics of Asian influenza isolates. Proc. Sot. Exptl. Biol. Med. 100,672-676. SETO, J. T., HICKEY, B. J., and RASMUSSEN, A. F., JR. (195913). Sialidase activity and related properties of influenza A2 viruses. Virology 9, 59% 611. TYRRELL, D. A. J., and VALENTINE, R. C. (1957). The assay of influenza virus particles by hemagglutination and electron microscopy. J. Gen. Microbial. 16,668-675. WILLIAMS, R. C., and BACKUS, R. C. (1949). Macromolecular weight determined by direct particle counting. J. Am. Chem. Sot. 71,4052-4057.