ANALYTICAL
BIOCHEMISTRY
Comparative
90,
273-288
(1978)
Measurements of Size and Polydispersity Several Insect Viruses
of
EGIDIJUS E. UZGIRIS,* DAVID H. CLUXTON,~ HORACE M. MAZZONES
AND
RALPH W. DEBLOIS,*
*General Electric Carporaie Research and Development, *Physics Department, Russell Sage College, Troy. Insect and Disease Laboratory. U.S. Department Connecticut 06574 Received
December
Schenectady. Net{% York 12301: Nrrz, York 12180; and $Forest of Agriculture. Hamden.
9, 1977
Comparative measurements of the sizes and polydispersity of the Tipula iridescent virus and the nuclear polyhedrosis viruses of the gypsy moth and European pine sawfly have been made by laser light-scattering spectroscopy, resistivepulse analysis, and electron microscopy. Size measurements are in substantial agreement by the three techniques. Polydisperse components in the various viral preparations are distinctly resolvable by resistive-pulse analysis. are measurable by electron microscopy, but are combined into a weighted average size by laser light-scattering spectroscopy.
The size of a virus is one parameter of value in its characterization. Electron microscopy is conventionally used for sizing viruses, but two other methods, laser light-scattering spectroscopy (1,2) and the resistivepulse technique (3-9, are also available and might show advantages in precision, resolution, and rapidity of measurement. Light-scattering spectroscopy has become widely used to measure the diffusion constants and hydrodynamic sizes of macromolecules and viruses. This method is well suited for measurements on pure monodisperse samples but is less suitable for the analysis of polydisperse samples. The resistive-pulse technique, on the other hand, can be readily used to size components in a complex mixture. This technique, therefore, may be particularly useful for studies in which polydispersity is naturally occurring and in which purification into single components is difficult to achieve. High-resolution sizing analysis of certain viruses by the resistive-pulse technique may also reveal structural variants that could not be observed readily by the other two techniques. To demonstrate these points, we have sized three insect viruses by all three techniques and compared the results. The viruses studied were (i) 273
0003-2697/78/0901-0273$02,00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
274
DEBLOIS
ET AL.
the Tipula iridescent nuclear polyhedrosis
virus (TIV)’ of the Tipula paludosa fly, and the viruses of (ii) the European pine sawfly (EPSV) (Neadiprion serrifer Geoffroy), and (iii) the gypsy moth (GMV) (Lymantria dispur Linnaeus). The Tipula iridescent virus is icosahedral in shape. The latter two are rod-shaped and are released from polyhedral inclusion bodies by dissolution in an alkaline solution. Electron microscopy has shown both rods and spheres in preparations of EPSV, with the spheres apparently resulting from bending of the rods in the dissolution process (6,7). Preparations of GMV show rods of polydisperse size. In addition to naked nucleocapsid rods, there are larger units consisting of varying numbers of such rods enclosed within single outer membranes (8- 11). Of special interest is how well this polydispersity can be analysed by the various techniques cited above. Another point of interest is the variability in size of virus particles among different preparations. MATERIALS Resistive-Pulse
AND METHODS
Technique
The Nanopar analyzer employs the resistive-pulse technique of the Coulter Counter and a pore of submicron diameter to size particles as they pass through one by one in fluid suspension. Details of the apparatus and its operation are given elsewhere (5). The following equations are used in determining particle sizes. In resistive-pulse analysis, spherical particles are measured with respect to standard latex spheres of diameter d, through the equation
where AE and AEo are the voltage-pulse amplitudes produced by particles of diameters d and d,, respectively, as they pass through the pore, and 9 is the diameter of the pore. If the particle is not a sphere, but may be approximated as an ellipsoid of revolution, then its volume V, may be readily determined in terms of the resistively equivalent diameter of Eq. [I] and a form factorf,, viz., V, = rd 3/4f,.
The form factorf,
is given by
fe = l-1 -n, ’ Abbreviations of the European
m
+ & i
used: TIV, Tip& iridescent pine sawfly; and GMV, nuclear
II
- -q 1-
rrl
coszp,
[31
virus; EPSV, nuclear polyhedrosis virus polyhedrosis virus of the gypsy moth.
INSECT
VIRUS
SIZE
AND
POLYDISPERSITY
275
where n, and n,, are extensively tabulated demagnetization factors for a field applied perpendicular and parallel, respectively, to the axis of revolution, and j3 is the angle between the axis of revolution and the field (12,13). It is observed experimentally that nonspherical particles generally pass through a pore with the long axis parallel to the axis of the pore, so that f, varies from 1.5 for a sphere to 1 for a long rod. The approximate shapes in a polydisperse sample are obtained through electron microscopy. Light
Scuttrring
The light-scattering spectrometer used was a standard arrangement consisting of a Spectra-Physics 15-mW He-Ne laser, an RCA No. 7265 photomultiplier, and a Princeton Applied Research model 101 A autocorrelation computer. Either the power spectrum or its Fourier transform, the autocorrelation function, of the scattered-light intensity fluctuations can be measured to yield information about the diffusion constant of the scattering particles. Technique has been well described in the literature (1.2). and we give only a brief review below. For a set of homogeneous particles the scattered-light intensity autocorrelation function is e-2DK2T,where T is the time delay, D is the diffusion constant, and K is the optical-scattering vector. Hence, light-intensity fluctuations decay away exponentially with a time constant of (2DK2)-‘. This decay can be measured precisely with the autocorrelation computer. By use ofthe Stokes-Einstein equation, D = kT/6rr,r, we can deduce the hydrodynamic size of the particles in question. Here k is Boltzmann’s constant. 7 is the vicosity, and r is the radius of the equivalent hydrodynamic sphere. If the particles are not homogeneous in size then this simple picture is considerably complicated by the fact that the autocorrelation function becomes a sum of exponentials. Such a function cannot be deconvoluted urliquely, especially when experimental data with limited signal to noise ratio are used (14,15). One can get some measure of the heterogeneity by noting to what degree the logarithm of the autocorrelation function departs from a straight line when plotted against time delay (16,17). But for small scale polydispersity involving several multiplets the curvature may be smaller than the scatter in the data owing to a finite signal to noise ratio. This in fact is the result for the virus samples studied in this work. For each set of virus measurements a calibration of the spectrometer and of the Nanopar analyzer was performed by measuring the hydrodynamic size of standard particles (91 or 109 nm polystyrene latex spheres, Lots LS- 1132-B and 2G3 W, respectively, Dow Chemical Co., Indianapolis. Indiana).
276
DEBLOIS
ET AI!,.
Electron Microscopy
Samples of the viruses suspended in water were prepared for viewing by transmission electron microscopy (Siemens Elmiskop 101 at 80 kV) by placing a drop of suspension on a lacy carbon film substrate and negatively staining with 1% uranyl acetate. Enlarged prints on paper were made from the photographic plates, resulting in a magnification of 150,000X or 300,000X. To avoid possible increase in the number of aggregated viruses, the virus was not concentrated before preparation for electron microscopy. The calibration of the magnification for the viruses is presumed to be the same as for careful measurements on negatively stained polystyrene latex spheres carried out at nearly the same time. For the sphere measurements, internal calibrations were performed with both diffraction grating replicas and catalase crystals (18). Measurements of 180 spheres of nominal 91 nm diameter in 13 photographs were plotted as a histogram and the mean diameter was found to be 79 ? 6 nm. A similar study of nominally 109-nm polystyrene spheres yields a value of 96 2 6 nm for a sample of 300 spheres in 11 photographs. Virus Preparations
The rod fractions from the nuclear polyhedrosis viruses of the gypsy moth and the European pine sawfly were prepared according to Bergold’s method (8). Viral inclusion bodies from each insect, purified by zonal rotor centrifugation (7,19), were lysed in a solution of 0.008 M Na&030.05 M NaCl, pH 10.5. The inclusion-body protein degraded to subunit size, freeing the rods contained within. Differential centrifugation, employing two cycles of speeds, 16OOg/5 min and 41,OOOg/45 min separated the rods (sedimented at the higher speed) from debris (sedimented at the lower speed) and from subunit inclusion-body protein (remained in solution at either speed). The rod pellet was resuspended in water after each sedimentation. A purified preparation of the Tipufa iridescent virus was obtained from Dr. A. Dodds (Connecticut Agricultural Experiment Station, New Haven, Connecticut). RESULTS Resistive-Pulse Measurements
Figure 1 is a histogram of number N versus pulse height for a mixture of the Tipula iridescent virus with 162-nm-diameter latex spheres (Lot 2F5X, nominally 176 nm in diameter, Dow Chemical Co., Indianapolis, Indiana). From the pulse peak positions we find a diameter for the virus of 180 nm, by use of Eq. [I] and a pore diameter of 550 nm. The above run plus five others give an average peak diameter of 180.5 ? 0.5 nm. The lower side peak visible here at pulse height 53 appears on all histograms and yields a diameter of 176.6 + 0.9 nm. This result
277
INSECT VIRUS SIZE AND POLYDISPERSITY
125 TIPULA IRIDESCENT VIRUS 180nm 100
25
0 IO
20
30
40
50
60
70
PULSE HEIGHT FIG. 1. Histogram of number N versus pulse height for a mixture of T&la iridescent virus and 162-nm-diameter polystyrene latex spheres yielding a diameter of 180 nm for the virus. The histogram was obtained for a mixture of 4 yl of the virus preparation and 0.05 ~1 of the sphere suspension with 1 ml of a KCl, Triton X-155 nonionic surfactant (Rohm & Haas, Philadelphia, Pennsylvania), Surfynol TG nonionic surfactant (Air Products and Chemicals, Inc., Allentown, Pennsylvania) solution. The suspension flowed through a 550~nmdiameter pore, 2.10 pm long, for 360 s at 0.6 in. of water pressure, with 1.200 V across the pore and 129 nA passing through it.
presumably represents a structural variant. The above diameters refer to those of the resistively equivalent spheres and should fall between the apex-to-apex and side-to-side dimensions of this icosahedral virus. Gypsy moth viruses display several size components in the pulse-height
278
DEBLOIS
ET AL.
300 GYPSYMOTHVIRUS
250 200 N 150 100 50 0
I
1
I
ML
IO 20 30 40 50 60 70 80 90 100 PULSE HEIGHT
FIG. 2. Histogram of number N versus pulse height for a preparation of gypsy moth virus, showing components of several sizes. The histogram was obtained for a mixture of 0.20 ml of a suspension of the virus, with 4 ml of tissue culture medium (RPM1 1640) containing 8% fetal calf serum, and 80 ~1 of Tween 80 nonionic surfactant (Atlas Chemical Industries, Inc., Wilmington, Delaware). The mixture was passed through a 0.2~pm Nuclepore filter. (The medium was one that happened to be readily available and that works well. Solutions of KC1 with surfactants may also be satisfactory, though the one used for TIV appears to disrupt GMV.) The viruses flowed electrokinetically through a pore of diameter 402 nm and length 2.31 pm for 565 s with 1.200 V and zero pressure across the pore and 83.5 nA flowing through it.
INSECT VIRUS SIZE AND POLYDISPERSITY
279
histogram of Fig. 2. Electron microscopy has revealed that varying numbers o f rods (nucleocapsids) of the nuclear polyhedrosis viruses may be enveloped in one outer membrane (3,4,11,14). With this evidence we may assign the numbered peaks to correspond to packets of one through four rods. Size measurements were made through a similar run with the viruses mixed with standard 234-nm polystyrene latex spheres (Lot 2G6S, Dow Chemical Co., Indianapolis, Indiana) that we take from our measurements to be 217 nm in diameter. Peak one, corresponding to single enveloped rods, yielded a resistively equivalent diameter of 152.4 nm. T w o later runs yielded 147.8 and 151.0 nm, for an average of 150.4 nm. Our observations by electron microscopy give this c o m p o n e n t an axial ratio of about 3.7 to 1. If we approximate the virus as an ellipsoid of revolution with its long axis parallel to the axis of the pore, then the demagnetizing factor n n is found to be 0.083, the form factor fe of Eq. [3] becomes 1.09, and the volume, through Eq. [2], is 2.45 x 10-1'~cma. This volume is equal to that of a sphere with a diameter of 167.3 nm, 11% larger than the resistively equivalent diameter. The peaks in Fig. 2 corresponding to two, three, and four rods enveloped in single outer membranes had resistively equivalent diameters o f 179.0, 196.1, and 210.8 nm, respectively. For estimated axial ratios of 3.1, 2.6, and 2.2, respectively, these correspond to volumes of 4.0, 5.1, and 6.1 x 10-15cm 3. The histogram of Fig. 3 for gypsy moth virus was made using a pore of diameter 309 nm, smaller than the one of 402-nm diameter used above. This has permitted resolution of single unenveloped rods, which appear in peak zero in the figure. (The left-hand peak of Fig. 2 does not correspond accurately to the unenveloped rods because of background noise and a high threshold setting for the analyzer.) Comparison with the peak position for 162-nm-diameter latex spheres mixed later with the virus yielded a resistively equivalent diameter of 94.5 nm. Combining this result with several other runs on several samples, we find a diameter of 93 + 3 nm. For an ellipsoidal axial ratio o f 8 to 1 we calculate a volume of(0.61 _+ 0.06) × 10-1~cm3. An alternative approximation as a cylinder, which would match the actual shape more closely but which is analyzable with less precision, gives a volume of not more than 0.63 × I0-1~cm3. Thus we may take the volume o f the unenveloped rod to be (0.62 _+ 0.07) × 10-15cm 3. This is equivalent in volume to a sphere of diameter 106 ~ 4 nm. Peaks 1, 2, and 3 of Fig. 3 yield resistively equivalent diameters of 140.1, 167.4, and 184.4 nm, respectively. These are smaller than the diameters for the corresponding peaks of Fig. 2 but are for a different sample. Combining results on three samples, we find a resistively equivalent diameter of 144 _+ 7 nm and a volume of (2.15 +_ 0.4) x 10-1~cm3 for the single enveloped rod. The error in the volume includes an allowance for uncertainty in the axial ratio.
280
DEBLOIS
ET AL.
200 L
GYPSY MOTH VIRUS 150 N
1 loo
50
0
IO 20 30 40 50 60 70 80 90 100 PULSE HEIGHT
FIG. 3. Histogram of number N versus pulse height for a preparation of gypsy moth virus, showing components corresponding to the unenveloped nucleocapsid rod (peak 0) and to one, two, and three rods enveloped in single outer membranes (peaks I, 2, and 3). The preparation consisted of 0.20 ml of a suspension of the virus, 7 ml of tissue culture medium with 8% fetal calf serum, and 40 ~1 of Tween 80. and was filtered through a 0.2 @m Nuclepore membrane. The viruses flowed through a pore of 309 nm diameter and length 2.22 pm for 390 s, with 1.00 V and 10 in. of water pressure across the pore and 44.1 nA passing through it.
A preparation of the virus of the European pine sawfly shows two peaks in the pulse-height histogram of Fig. 4. The resistively equivalent diameters of 112.9 and 123.8 nm for the two components are calibrated against standard 109-nm latex spheres, taken to be 103 nm in diameter from our measurements, that were subsequently added to the sample suspension. A histogram taken with the added spheres minus the above histogram, duly corrected for a difference in running times, produced the peak shown for the spheres.
INSECT
VIRUS
SIZE
AND
POLYDISPERSITY
281
Electron microscopy of the preparation showed a mixture of rods and globules. If the globules are enveloped rods that have been bent into spherical forms by the dissolution process (6,7), then this would indicate that the lower peak corresponds to the rods and the higher peak to the globules. If the rod is approximated as a prolate ellipsoid of axial ratio 10 to 3, with its long axis parallel to the axis of the pore, then its volume is found by Eqs. [2] and [3] to be 1.02 x lo-15cm3. If the globule is taken as a sphere, then its volume is 0.99 x IO-‘“cm3, in close agreement with that for the rod. With two other runs on the above lot of viruses and two on another preparation, we found average resistively equivalent peak diameters of 113 i 1 and I23 + 2 nm, yielding volumes of (1.02 t 0.03) x IO-15cm3 and (0.97 * 0.05) x IO-%rn” for the rod and globule components, respectively. Uncertainty about the actual shapes of the components might double the probable errors of these determinations. The unenveloped rod of the European pine sawfly virus is not distinctly resolvable in the histogram of Fig. 4 in the counts at low pulse height. However, runs on two other lots do show peaks or side peaks above debris that are distinct enough to assign to this rod type and that give it a resistively equivalent diameter of 86 2 4 nm. Using an axial ratio of 5.7 ? 1 to 1, as determined by electron microscopy, we calculate a volume of (0.48 i 0.08) x lo-15cm3 and a spherically equivalent diameter of 97 5 6 nm. Hydrodynamic
Size Measurements
Using Light-Scattering
Spectroscopy
Measurements on Dow Chemical Co. polystyrene latex spheres in water under many conditions of alignment and temperature gave the hydrodynamic diameter of the nominally 91-nm spheres (Lot LS-1132-B) as 84 & 2 nm. All of the resistive-pulse measurements were calibrated in terms of this standard. For instance, the relative size ratio of 109 to 91-nm nominal size spheres was determined by resistive-pulse measurement to be 1.23. Hence, the absolute size in terms of the standard was a 103 r 3-nm diameter, which compares to 101 c 3 nm by light scattering determinations and to 96 & 6 nm by electron microscopy. Similar relative size measurements yielded 114 nm for nominally 126-nm spheres (Lot LS-052-A), 162 nm for nominally 176-nm spheres (Lot 2F5X), and 217 nm for nominally 234-nm spheres (Lot 2G6S). The results for the viruses are listed in Table 1, with the indicated uncertainties representing both instrumental and sample variations. For light scattering, only one number is given, which is biased toward the larger sizes present owing to the rapid increase of scattered intensity with particle radius. The samples as measured had concentrations of nominally 1O’Oto 10” particles per milliliter. Runs with diluted samples over a range of at least one order of magnitude indicated no measurable effect of concentration on effective size. Also, dilutions of the virus with 0.005 N NaCl solutions did not lead to any difference in measured size.
282
DEBLOIS
ET AL.
500 450
EUROPEAN PINE SAWFLY VIRUS
+
400 I
350 -c
E M -
300 N 250
I E c
200
4
G
11r
150 100 50 0
I ,
I
20
L
I
30
I
I
I
40 50 60 PULSE HEIGHT
I
70
INSECT
VIRUS
SIZE
AND
TABLE SUMMARY Resistive
Particle volume (x IO” cm7
VINS
1
OF MEASUREMENTS pulse
Electron
Equiv. sphere diameter
lnm)
(x
IO’”
Light scattering
microscopy
Particle volume
(iv
Gypsy moth virus Unenveloped rod (axial ratio 8 to 1) Enveloped rod (axial ratio 3.7 t0 I)
283
POLYDISPERSITY
cm31
Equiv. sphere diameter
Particle volume
(nm)
Equiv. sphere diameter
(X IO’S Crn~)
(nm)
2.9 k 0.4
176 2 9
1.05 + 0.16
126 2 6
5.0 + 0.8
212 -t 10
= 461
0.62 f 0.07
I06 f 4
0.50 f 0.14
98 ? 9
2.15 t 0.4
I60 e 9
3.1 f 0.7
181 + I4
(N = 59) European pine sawfly virus Unenveloped rod (axial ratio 5.7 t0 I) Globule (curled-up enveloped rod) Enveloped rod (axial ratio 3.3 to I)
0.48 T 0.08
97 -t 6
0.48 2 0. I6
97 t
II
0.97 r 0. IO
123 2 4
1.02 f 0.16
I25 2 7
1.02 z 0.06
I25 + 3
1.08 2 0.25
127 i
IO
(N = 21) iridescent Wxahedront
Tipuia
virus 3.07 + 0.02
180.5 I 0.5
2.6 i 0.1
170 2 2
Although the viruses are rod-shaped (except for TIV), we see no appreciable effects of rotational motion, since the rotational contribution to the scattered wave is small at 90” for this size of particle (20). This conclusion is supported by measurements of the diffusion constant at 60 which yielded values not significantly different from those at 90”. For gypsy moth virus and the European pine sawfly virus, the sizes shown in Table 1 for the equivalent sphere are determined from the hydrodynamic diameter by including a friction factor for diffusion of an ellipsoid in solution (21). The friction factors vary with axial ratios of the rods (obtained from the electron micrographs). The particle volumes quoted in Table 1 are calculated from V, = 4m”/3, where r is the radius of the equivalent sphere. Comparison of these values with electron micrograph and resistive-pulse measurement results can only be approximate, since different components of the polydisperse samples have different FIG. 4. Histogram of number N versus pulse height for a preparation of European pine sawfly virus, showing two components. The dashed lines show the histogram obtained for 103-nm-diameter polystyrene latex spheres added subsequently to the virus. The sample consisted of 0.40 ml of the preparation of viruses mixed with 4 ml of RPM1 1640 nutrient medium containing 8% fetal calf serum and 0.06 ml of Tween 80. The mixture was filtered with 0.2~pm Nuclepore. The suspension of viruses flowed through a 3IPnm-diameter pore, 2.58 pm long. for 297 s at 9 in of water pressure. with 1.000 V across the pore and 38.0 nA passing through it.
284
DEBLOIS
ET AL.
axial ratios and thus different friction factors, and the light scattering is not linear with particle size, but varies as approximately r4 in this size range. For the Tipula iridescent virus, the measured hydrodynamic diameters are given without further corrections because, for comparison, the icosahedral dimensions of the virus, i.e., face-to-face and apex-to-apex distances, were not clearly discerned in the electron micrographs and only some average spherical diameter was obtained. The results clearly indicate that the hydrodynamic size is much larger than the virus size determined either by electron microscopy or by resistive-pulse analysis. We had taken precautions to eliminate debris and virus aggregates from the samples by filtering and by adding surfactant. One explanation for the observed discrepancy is that there may be a peripheral fuzzy coat surrounding the TIV, as reported by Stoltz (22). Such an amorphous outer layer would have the most effect on hydrodynamic properties of the virus and would have relatively little effect on the resistive-pulse determination because the conductivity of the fuzzy region would not differ very much from the conductivity of the surrounding fluid. The fuzz was not visible in our micrographs, but it is apparently of such low density that it is not visible using ordinary techniques. Near the end of this investigation a photon-counting system was completed. The results obtained with this system verified the earlier measurements on the polystyrene spheres as well as the virus samples. Figure 5 is an example of the light-scattering data showing the 100 channels of output on a semilog plot for gypsy moth virus. Above the data points is shown an autocorrelation function that was generated numericahy by using the multiplet distribution determined in Fig. 3, the appropriate diffusion coefficient for each component, and a lightscattering weighting factor of r4.3 as an approximation to the departure from true Rayleigh scattering for particles of this size (15). The numerical simulation of the correlation function based on the resistive-pulse data gives a straight line for the logarithm of the autocorrelation function. The slope of this line is close to the experimentally observed slope. Furthermore, the curvature in the numerically generated function is far less than the scatter in the experimental data. Thus, the further analysis of light-scattering data by expansions into cumulants was not useful in these cases. Other workers studying viruses have also come to this conclusion regarding cumulant analysis with limited signal to noise conditions (15). Electron Microscopy
Figure 6 shows typical micrographs of the three types of viruses studied. The rod viruses appear as right circular cylinders with hemispherical
INSECT VIRUS SIZE AND POLYDISPERSITY
285
GMV
FIG. 5. The autocorrelation function of laser light scattered at 90” (homodyne) from gypsy moth virus in water at room temperature. Tau is the delay time between successive samplings of the light intensity. The dots in the lower line are the experimental data. The solid line connects points numerically calculated from the size distribution determined in Fig. 3. A weighting factor of r4.3 was used to approximate the variation in optical cross section as a function of size. Diffusion constants for each multiplet component corresponded to the volume and the friction factor of that component. The upper curve is displaced to the right by 0.2 ms units for clarity.
end caps (23). For a cylindrical radius b and total length of 2a, the volume of such particles is V = 2&+‘a(l-b/3a). The results of the volume measurements from the electron micrographs are indicated in Table 1. Histograms of axial ratios were also established and these were used to correct for shape in determining equivalent spherical diameters both for the resistive-pulse measurements and for the light-scattering measurements. The number of enveloped GMV rods counted was only 16 and for this reason no multiplet structure could be observed in particle number vs. volume. In the European pine sawfly virus preparations, we counted 20 spherical globules among the virus rods. These are presumed to be curled-up enveloped rods (6,7).
286
DEBLOIS
ET AL.
INSECT
VIRUS
SIZE
AND
POLYDISPERSITY
287
Tipuln iridescent viruses were photographed at 300,000x magnification and were basically round in cross section with hints of flat-sidedness as evidence of their icosahedral shape (Fig. 6). A histogram of 21 viruses yielded a sharp peak representing a volume of (2.6 2 0. I) x IO-l5 cm3 from the average measured diameter of 170 + 2 nm. A much smaller peak representing only seven viruses indicated a possible secondary group with a diameter of 162 & 4 nm. The results in Table 1 indicate substantial agreement between the measurements of size by resistive-pulse, light-scattering, and electron microscope techniques. The first two methods measure hydrated virus size while the last measures the dehydrated virus size. The data of Table 1 show no significant shrinkage in size due to dehydration for the GMV and EPSV unencapsulated rods. For the encapsulated rods of GMV the electron microscope measurements were too few in number to resolve the multiplets; thus an accurate comparison with the resistive-pulse values could not be made. Dehydration may produce a small shrinkage in Tip& iridescent virus but does not appear to affect the encapsulated EPSV rods. Separate components of the polydisperse virus preparations could not be resolved by laser light scattering and these measurements were heavily biased toward the largest components present in a sample, as expected from Mie scattering theory (15). The light-scattering measurements for Tipulu iridescent virus show the largest departure from the other two methods, possibly because of a fuzzy, amorphous outer coat. The electron microscopy and resistive-pulse sizes for TIV are in basic agreement if one takes into account the spread in sizes visible in the electron micrographs. An important result of this investigation is that the Nanopar analyzer, using the resistive-pulse technique, is able to resolve distinctly the various components of a polydisperse virus sample and to measure their absolute volumes when the shapes are known. Data are gathered with greater ease than by electron microscopy and with probably greater accuracy, since preparation for electron microscopy may lead to particle distortion. Thus, the analyzer may be especially suitable for characterizing or classifying polydisperse viruses. Virus sizes in a given run are measurable to several nanometers or better. The smallest virus sized thus far is 59 nm in diameter (24). Signalto-noise observations indicate that viruses of half that diameter could be sized under special conditions of cleanliness, high electrolyte concentration, and small pore diameter. Our measurements by resistive-pulse analysis and electron microscopy agree with previous observations of two shapes of virus particles, rodshaped and globular, in some preparations of the European pine sawfly virus, and of varying numbers of virus rods enveloped in single outer membranes for the gypsy moth virus. In addition, these techniques may
DEBLOIS
288
have sized out a structural virus.
ET At.
variant in a preparation
of Tipula iridescent
ACKNOWLEDGMENTS We thank R. R. Russell for the electron microscopy and M. V. Doyle for analytical help.
REFERENCES 1. Carlson, F. D. (1975) Annu. Rev. Biophys. Bioeng. 4, 243-264. 2. Cummins, H. Z., and Swinney, H. L. (1970) in Progress in Optics (Wolf, E.. ed.), Vol. 8, pp. 135-200, North-Holland, Amsterdam. 3. DeBlois, R. W., and Bean, C. P. (1970) Rev. Sci. lnstrum. 41, 909-916. 4. DeBlois, R. W., and Wesley, R. K. A. (1977) .I. Viral. 23, 227-233. 5. DeBlois, R. W., Bean, C. P., and Wesley, R. K. A. (1977) J. Collvid Interface Sci. 61, 323-335. 6. Bahr. G. F., Engler, W. F., and Mazzone. H. M. (1976) Quart. Rev. Biophys. 9, 459-489. 7. Mazzone, H. M., Breillatt, J. P., and Anderson, N. G. (1970) in Proceedings of the IVth International Colloquium on Insect Pathology, pp. 371-379, College Park, Maryland. 8. Bergold, G. H. (1953) in Advances in Virus Research. (Smith, K. M., and Lauffer. M. A., eds.) Vol. 1, pp. 91-139, Academic Press, New York. 9. Bergold, G. H. (1963) in Insect Pathology (Steinhaus, E. A., ed.). Vol. 1 pp. 413456. Academic Press, New York. 10. Harrap, K. A. (1972) Virology 50, 133-139. 11. Mazzone, H. M., Breillatt, J. P., and Bahr, G. F. (1973) in Fifth International Colloquium on Insect Pathology and Microbial Control, p. 42, Oxford University. 12. Osbom, J. A. (1945) Phys. Rev. 67, 351-357. 13. Stoner, E. C. (1945)Phil. Mag. 36, 803-821. 14. Pusey, P. N., Koppel, D. E., Schaefer, D. W., Camerini-Otero, R. D.. and Koenig. S. H. (1974) Biochemistry 13, 952-960. 15. Salmeen, I., Rimai, L., Luftig, R. B., Liebes, L., Retzel, E., Rich, M., and McCormick, J. J. (1976) J. Virol. 17, 584-596. 16. Bargeron, C. B. (1974) J. Chem. Phys. 60, 2516-2519. 17. Koppel, D. E. (1972)J. Chem. Phys. 57, 4814-4820. 18. Wrigley, N. G. (1968) J. Utrastruct. Res. 24, 589-590. 19. Breillatt, J. P., Brantley. J. N., Mazzone. H. M., Martignoni, M. E., Franklin, J. E., and Anderson, N. G. (1972) Appl. Microbial. 23, 923-930. 20. Pecora, R. (1965) J. Chem. Phys. 49, 1036-1043. 21. Tanford, C. (1961) Physic& Chemistry of Macromolecules, pp. 324-328, Wiley, New York. 22. Stoltz, D. B. (1971) J. Ultrastruct. Res. 37, 219-239. 23. Mazzone, H. M., and Tignov, G. H. (1976) in Advances in Virus Research, Vol. 20, pp. 237-270, Academic Press. New York. 24. Feuer, B., Uzgiris, E. E., DeBlois, R. W.. Cluxton, D. H., and Lenard, J. (1978) Biophys. J. 21, 38a.
BIOCHEMISTRY
Comparative
90,
273-288
(1978)
Measurements of Size and Polydispersity Several Insect Viruses
of
EGIDIJUS E. UZGIRIS,* DAVID H. CLUXTON,~ HORACE M. MAZZONES
AND
RALPH W. DEBLOIS,*
*General Electric Carporaie Research and Development, *Physics Department, Russell Sage College, Troy. Insect and Disease Laboratory. U.S. Department Connecticut 06574 Received
December
Schenectady. Net{% York 12301: Nrrz, York 12180; and $Forest of Agriculture. Hamden.
9, 1977
Comparative measurements of the sizes and polydispersity of the Tipula iridescent virus and the nuclear polyhedrosis viruses of the gypsy moth and European pine sawfly have been made by laser light-scattering spectroscopy, resistivepulse analysis, and electron microscopy. Size measurements are in substantial agreement by the three techniques. Polydisperse components in the various viral preparations are distinctly resolvable by resistive-pulse analysis. are measurable by electron microscopy, but are combined into a weighted average size by laser light-scattering spectroscopy.
The size of a virus is one parameter of value in its characterization. Electron microscopy is conventionally used for sizing viruses, but two other methods, laser light-scattering spectroscopy (1,2) and the resistivepulse technique (3-9, are also available and might show advantages in precision, resolution, and rapidity of measurement. Light-scattering spectroscopy has become widely used to measure the diffusion constants and hydrodynamic sizes of macromolecules and viruses. This method is well suited for measurements on pure monodisperse samples but is less suitable for the analysis of polydisperse samples. The resistive-pulse technique, on the other hand, can be readily used to size components in a complex mixture. This technique, therefore, may be particularly useful for studies in which polydispersity is naturally occurring and in which purification into single components is difficult to achieve. High-resolution sizing analysis of certain viruses by the resistive-pulse technique may also reveal structural variants that could not be observed readily by the other two techniques. To demonstrate these points, we have sized three insect viruses by all three techniques and compared the results. The viruses studied were (i) 273
0003-2697/78/0901-0273$02,00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
274
DEBLOIS
ET AL.
the Tipula iridescent nuclear polyhedrosis
virus (TIV)’ of the Tipula paludosa fly, and the viruses of (ii) the European pine sawfly (EPSV) (Neadiprion serrifer Geoffroy), and (iii) the gypsy moth (GMV) (Lymantria dispur Linnaeus). The Tipula iridescent virus is icosahedral in shape. The latter two are rod-shaped and are released from polyhedral inclusion bodies by dissolution in an alkaline solution. Electron microscopy has shown both rods and spheres in preparations of EPSV, with the spheres apparently resulting from bending of the rods in the dissolution process (6,7). Preparations of GMV show rods of polydisperse size. In addition to naked nucleocapsid rods, there are larger units consisting of varying numbers of such rods enclosed within single outer membranes (8- 11). Of special interest is how well this polydispersity can be analysed by the various techniques cited above. Another point of interest is the variability in size of virus particles among different preparations. MATERIALS Resistive-Pulse
AND METHODS
Technique
The Nanopar analyzer employs the resistive-pulse technique of the Coulter Counter and a pore of submicron diameter to size particles as they pass through one by one in fluid suspension. Details of the apparatus and its operation are given elsewhere (5). The following equations are used in determining particle sizes. In resistive-pulse analysis, spherical particles are measured with respect to standard latex spheres of diameter d, through the equation
where AE and AEo are the voltage-pulse amplitudes produced by particles of diameters d and d,, respectively, as they pass through the pore, and 9 is the diameter of the pore. If the particle is not a sphere, but may be approximated as an ellipsoid of revolution, then its volume V, may be readily determined in terms of the resistively equivalent diameter of Eq. [I] and a form factorf,, viz., V, = rd 3/4f,.
The form factorf,
is given by
fe = l-1 -n, ’ Abbreviations of the European
m
+ & i
used: TIV, Tip& iridescent pine sawfly; and GMV, nuclear
II
- -q 1-
rrl
coszp,
[31
virus; EPSV, nuclear polyhedrosis virus polyhedrosis virus of the gypsy moth.
INSECT
VIRUS
SIZE
AND
POLYDISPERSITY
275
where n, and n,, are extensively tabulated demagnetization factors for a field applied perpendicular and parallel, respectively, to the axis of revolution, and j3 is the angle between the axis of revolution and the field (12,13). It is observed experimentally that nonspherical particles generally pass through a pore with the long axis parallel to the axis of the pore, so that f, varies from 1.5 for a sphere to 1 for a long rod. The approximate shapes in a polydisperse sample are obtained through electron microscopy. Light
Scuttrring
The light-scattering spectrometer used was a standard arrangement consisting of a Spectra-Physics 15-mW He-Ne laser, an RCA No. 7265 photomultiplier, and a Princeton Applied Research model 101 A autocorrelation computer. Either the power spectrum or its Fourier transform, the autocorrelation function, of the scattered-light intensity fluctuations can be measured to yield information about the diffusion constant of the scattering particles. Technique has been well described in the literature (1.2). and we give only a brief review below. For a set of homogeneous particles the scattered-light intensity autocorrelation function is e-2DK2T,where T is the time delay, D is the diffusion constant, and K is the optical-scattering vector. Hence, light-intensity fluctuations decay away exponentially with a time constant of (2DK2)-‘. This decay can be measured precisely with the autocorrelation computer. By use ofthe Stokes-Einstein equation, D = kT/6rr,r, we can deduce the hydrodynamic size of the particles in question. Here k is Boltzmann’s constant. 7 is the vicosity, and r is the radius of the equivalent hydrodynamic sphere. If the particles are not homogeneous in size then this simple picture is considerably complicated by the fact that the autocorrelation function becomes a sum of exponentials. Such a function cannot be deconvoluted urliquely, especially when experimental data with limited signal to noise ratio are used (14,15). One can get some measure of the heterogeneity by noting to what degree the logarithm of the autocorrelation function departs from a straight line when plotted against time delay (16,17). But for small scale polydispersity involving several multiplets the curvature may be smaller than the scatter in the data owing to a finite signal to noise ratio. This in fact is the result for the virus samples studied in this work. For each set of virus measurements a calibration of the spectrometer and of the Nanopar analyzer was performed by measuring the hydrodynamic size of standard particles (91 or 109 nm polystyrene latex spheres, Lots LS- 1132-B and 2G3 W, respectively, Dow Chemical Co., Indianapolis. Indiana).
276
DEBLOIS
ET AI!,.
Electron Microscopy
Samples of the viruses suspended in water were prepared for viewing by transmission electron microscopy (Siemens Elmiskop 101 at 80 kV) by placing a drop of suspension on a lacy carbon film substrate and negatively staining with 1% uranyl acetate. Enlarged prints on paper were made from the photographic plates, resulting in a magnification of 150,000X or 300,000X. To avoid possible increase in the number of aggregated viruses, the virus was not concentrated before preparation for electron microscopy. The calibration of the magnification for the viruses is presumed to be the same as for careful measurements on negatively stained polystyrene latex spheres carried out at nearly the same time. For the sphere measurements, internal calibrations were performed with both diffraction grating replicas and catalase crystals (18). Measurements of 180 spheres of nominal 91 nm diameter in 13 photographs were plotted as a histogram and the mean diameter was found to be 79 ? 6 nm. A similar study of nominally 109-nm polystyrene spheres yields a value of 96 2 6 nm for a sample of 300 spheres in 11 photographs. Virus Preparations
The rod fractions from the nuclear polyhedrosis viruses of the gypsy moth and the European pine sawfly were prepared according to Bergold’s method (8). Viral inclusion bodies from each insect, purified by zonal rotor centrifugation (7,19), were lysed in a solution of 0.008 M Na&030.05 M NaCl, pH 10.5. The inclusion-body protein degraded to subunit size, freeing the rods contained within. Differential centrifugation, employing two cycles of speeds, 16OOg/5 min and 41,OOOg/45 min separated the rods (sedimented at the higher speed) from debris (sedimented at the lower speed) and from subunit inclusion-body protein (remained in solution at either speed). The rod pellet was resuspended in water after each sedimentation. A purified preparation of the Tipufa iridescent virus was obtained from Dr. A. Dodds (Connecticut Agricultural Experiment Station, New Haven, Connecticut). RESULTS Resistive-Pulse Measurements
Figure 1 is a histogram of number N versus pulse height for a mixture of the Tipula iridescent virus with 162-nm-diameter latex spheres (Lot 2F5X, nominally 176 nm in diameter, Dow Chemical Co., Indianapolis, Indiana). From the pulse peak positions we find a diameter for the virus of 180 nm, by use of Eq. [I] and a pore diameter of 550 nm. The above run plus five others give an average peak diameter of 180.5 ? 0.5 nm. The lower side peak visible here at pulse height 53 appears on all histograms and yields a diameter of 176.6 + 0.9 nm. This result
277
INSECT VIRUS SIZE AND POLYDISPERSITY
125 TIPULA IRIDESCENT VIRUS 180nm 100
25
0 IO
20
30
40
50
60
70
PULSE HEIGHT FIG. 1. Histogram of number N versus pulse height for a mixture of T&la iridescent virus and 162-nm-diameter polystyrene latex spheres yielding a diameter of 180 nm for the virus. The histogram was obtained for a mixture of 4 yl of the virus preparation and 0.05 ~1 of the sphere suspension with 1 ml of a KCl, Triton X-155 nonionic surfactant (Rohm & Haas, Philadelphia, Pennsylvania), Surfynol TG nonionic surfactant (Air Products and Chemicals, Inc., Allentown, Pennsylvania) solution. The suspension flowed through a 550~nmdiameter pore, 2.10 pm long, for 360 s at 0.6 in. of water pressure, with 1.200 V across the pore and 129 nA passing through it.
presumably represents a structural variant. The above diameters refer to those of the resistively equivalent spheres and should fall between the apex-to-apex and side-to-side dimensions of this icosahedral virus. Gypsy moth viruses display several size components in the pulse-height
278
DEBLOIS
ET AL.
300 GYPSYMOTHVIRUS
250 200 N 150 100 50 0
I
1
I
ML
IO 20 30 40 50 60 70 80 90 100 PULSE HEIGHT
FIG. 2. Histogram of number N versus pulse height for a preparation of gypsy moth virus, showing components of several sizes. The histogram was obtained for a mixture of 0.20 ml of a suspension of the virus, with 4 ml of tissue culture medium (RPM1 1640) containing 8% fetal calf serum, and 80 ~1 of Tween 80 nonionic surfactant (Atlas Chemical Industries, Inc., Wilmington, Delaware). The mixture was passed through a 0.2~pm Nuclepore filter. (The medium was one that happened to be readily available and that works well. Solutions of KC1 with surfactants may also be satisfactory, though the one used for TIV appears to disrupt GMV.) The viruses flowed electrokinetically through a pore of diameter 402 nm and length 2.31 pm for 565 s with 1.200 V and zero pressure across the pore and 83.5 nA flowing through it.
INSECT VIRUS SIZE AND POLYDISPERSITY
279
histogram of Fig. 2. Electron microscopy has revealed that varying numbers o f rods (nucleocapsids) of the nuclear polyhedrosis viruses may be enveloped in one outer membrane (3,4,11,14). With this evidence we may assign the numbered peaks to correspond to packets of one through four rods. Size measurements were made through a similar run with the viruses mixed with standard 234-nm polystyrene latex spheres (Lot 2G6S, Dow Chemical Co., Indianapolis, Indiana) that we take from our measurements to be 217 nm in diameter. Peak one, corresponding to single enveloped rods, yielded a resistively equivalent diameter of 152.4 nm. T w o later runs yielded 147.8 and 151.0 nm, for an average of 150.4 nm. Our observations by electron microscopy give this c o m p o n e n t an axial ratio of about 3.7 to 1. If we approximate the virus as an ellipsoid of revolution with its long axis parallel to the axis of the pore, then the demagnetizing factor n n is found to be 0.083, the form factor fe of Eq. [3] becomes 1.09, and the volume, through Eq. [2], is 2.45 x 10-1'~cma. This volume is equal to that of a sphere with a diameter of 167.3 nm, 11% larger than the resistively equivalent diameter. The peaks in Fig. 2 corresponding to two, three, and four rods enveloped in single outer membranes had resistively equivalent diameters o f 179.0, 196.1, and 210.8 nm, respectively. For estimated axial ratios of 3.1, 2.6, and 2.2, respectively, these correspond to volumes of 4.0, 5.1, and 6.1 x 10-15cm 3. The histogram of Fig. 3 for gypsy moth virus was made using a pore of diameter 309 nm, smaller than the one of 402-nm diameter used above. This has permitted resolution of single unenveloped rods, which appear in peak zero in the figure. (The left-hand peak of Fig. 2 does not correspond accurately to the unenveloped rods because of background noise and a high threshold setting for the analyzer.) Comparison with the peak position for 162-nm-diameter latex spheres mixed later with the virus yielded a resistively equivalent diameter of 94.5 nm. Combining this result with several other runs on several samples, we find a diameter of 93 + 3 nm. For an ellipsoidal axial ratio o f 8 to 1 we calculate a volume of(0.61 _+ 0.06) × 10-1~cm3. An alternative approximation as a cylinder, which would match the actual shape more closely but which is analyzable with less precision, gives a volume of not more than 0.63 × I0-1~cm3. Thus we may take the volume o f the unenveloped rod to be (0.62 _+ 0.07) × 10-15cm 3. This is equivalent in volume to a sphere of diameter 106 ~ 4 nm. Peaks 1, 2, and 3 of Fig. 3 yield resistively equivalent diameters of 140.1, 167.4, and 184.4 nm, respectively. These are smaller than the diameters for the corresponding peaks of Fig. 2 but are for a different sample. Combining results on three samples, we find a resistively equivalent diameter of 144 _+ 7 nm and a volume of (2.15 +_ 0.4) x 10-1~cm3 for the single enveloped rod. The error in the volume includes an allowance for uncertainty in the axial ratio.
280
DEBLOIS
ET AL.
200 L
GYPSY MOTH VIRUS 150 N
1 loo
50
0
IO 20 30 40 50 60 70 80 90 100 PULSE HEIGHT
FIG. 3. Histogram of number N versus pulse height for a preparation of gypsy moth virus, showing components corresponding to the unenveloped nucleocapsid rod (peak 0) and to one, two, and three rods enveloped in single outer membranes (peaks I, 2, and 3). The preparation consisted of 0.20 ml of a suspension of the virus, 7 ml of tissue culture medium with 8% fetal calf serum, and 40 ~1 of Tween 80. and was filtered through a 0.2 @m Nuclepore membrane. The viruses flowed through a pore of 309 nm diameter and length 2.22 pm for 390 s, with 1.00 V and 10 in. of water pressure across the pore and 44.1 nA passing through it.
A preparation of the virus of the European pine sawfly shows two peaks in the pulse-height histogram of Fig. 4. The resistively equivalent diameters of 112.9 and 123.8 nm for the two components are calibrated against standard 109-nm latex spheres, taken to be 103 nm in diameter from our measurements, that were subsequently added to the sample suspension. A histogram taken with the added spheres minus the above histogram, duly corrected for a difference in running times, produced the peak shown for the spheres.
INSECT
VIRUS
SIZE
AND
POLYDISPERSITY
281
Electron microscopy of the preparation showed a mixture of rods and globules. If the globules are enveloped rods that have been bent into spherical forms by the dissolution process (6,7), then this would indicate that the lower peak corresponds to the rods and the higher peak to the globules. If the rod is approximated as a prolate ellipsoid of axial ratio 10 to 3, with its long axis parallel to the axis of the pore, then its volume is found by Eqs. [2] and [3] to be 1.02 x lo-15cm3. If the globule is taken as a sphere, then its volume is 0.99 x IO-‘“cm3, in close agreement with that for the rod. With two other runs on the above lot of viruses and two on another preparation, we found average resistively equivalent peak diameters of 113 i 1 and I23 + 2 nm, yielding volumes of (1.02 t 0.03) x IO-15cm3 and (0.97 * 0.05) x IO-%rn” for the rod and globule components, respectively. Uncertainty about the actual shapes of the components might double the probable errors of these determinations. The unenveloped rod of the European pine sawfly virus is not distinctly resolvable in the histogram of Fig. 4 in the counts at low pulse height. However, runs on two other lots do show peaks or side peaks above debris that are distinct enough to assign to this rod type and that give it a resistively equivalent diameter of 86 2 4 nm. Using an axial ratio of 5.7 ? 1 to 1, as determined by electron microscopy, we calculate a volume of (0.48 i 0.08) x lo-15cm3 and a spherically equivalent diameter of 97 5 6 nm. Hydrodynamic
Size Measurements
Using Light-Scattering
Spectroscopy
Measurements on Dow Chemical Co. polystyrene latex spheres in water under many conditions of alignment and temperature gave the hydrodynamic diameter of the nominally 91-nm spheres (Lot LS-1132-B) as 84 & 2 nm. All of the resistive-pulse measurements were calibrated in terms of this standard. For instance, the relative size ratio of 109 to 91-nm nominal size spheres was determined by resistive-pulse measurement to be 1.23. Hence, the absolute size in terms of the standard was a 103 r 3-nm diameter, which compares to 101 c 3 nm by light scattering determinations and to 96 & 6 nm by electron microscopy. Similar relative size measurements yielded 114 nm for nominally 126-nm spheres (Lot LS-052-A), 162 nm for nominally 176-nm spheres (Lot 2F5X), and 217 nm for nominally 234-nm spheres (Lot 2G6S). The results for the viruses are listed in Table 1, with the indicated uncertainties representing both instrumental and sample variations. For light scattering, only one number is given, which is biased toward the larger sizes present owing to the rapid increase of scattered intensity with particle radius. The samples as measured had concentrations of nominally 1O’Oto 10” particles per milliliter. Runs with diluted samples over a range of at least one order of magnitude indicated no measurable effect of concentration on effective size. Also, dilutions of the virus with 0.005 N NaCl solutions did not lead to any difference in measured size.
282
DEBLOIS
ET AL.
500 450
EUROPEAN PINE SAWFLY VIRUS
+
400 I
350 -c
E M -
300 N 250
I E c
200
4
G
11r
150 100 50 0
I ,
I
20
L
I
30
I
I
I
40 50 60 PULSE HEIGHT
I
70
INSECT
VIRUS
SIZE
AND
TABLE SUMMARY Resistive
Particle volume (x IO” cm7
VINS
1
OF MEASUREMENTS pulse
Electron
Equiv. sphere diameter
lnm)
(x
IO’”
Light scattering
microscopy
Particle volume
(iv
Gypsy moth virus Unenveloped rod (axial ratio 8 to 1) Enveloped rod (axial ratio 3.7 t0 I)
283
POLYDISPERSITY
cm31
Equiv. sphere diameter
Particle volume
(nm)
Equiv. sphere diameter
(X IO’S Crn~)
(nm)
2.9 k 0.4
176 2 9
1.05 + 0.16
126 2 6
5.0 + 0.8
212 -t 10
= 461
0.62 f 0.07
I06 f 4
0.50 f 0.14
98 ? 9
2.15 t 0.4
I60 e 9
3.1 f 0.7
181 + I4
(N = 59) European pine sawfly virus Unenveloped rod (axial ratio 5.7 t0 I) Globule (curled-up enveloped rod) Enveloped rod (axial ratio 3.3 to I)
0.48 T 0.08
97 -t 6
0.48 2 0. I6
97 t
II
0.97 r 0. IO
123 2 4
1.02 f 0.16
I25 2 7
1.02 z 0.06
I25 + 3
1.08 2 0.25
127 i
IO
(N = 21) iridescent Wxahedront
Tipuia
virus 3.07 + 0.02
180.5 I 0.5
2.6 i 0.1
170 2 2
Although the viruses are rod-shaped (except for TIV), we see no appreciable effects of rotational motion, since the rotational contribution to the scattered wave is small at 90” for this size of particle (20). This conclusion is supported by measurements of the diffusion constant at 60 which yielded values not significantly different from those at 90”. For gypsy moth virus and the European pine sawfly virus, the sizes shown in Table 1 for the equivalent sphere are determined from the hydrodynamic diameter by including a friction factor for diffusion of an ellipsoid in solution (21). The friction factors vary with axial ratios of the rods (obtained from the electron micrographs). The particle volumes quoted in Table 1 are calculated from V, = 4m”/3, where r is the radius of the equivalent sphere. Comparison of these values with electron micrograph and resistive-pulse measurement results can only be approximate, since different components of the polydisperse samples have different FIG. 4. Histogram of number N versus pulse height for a preparation of European pine sawfly virus, showing two components. The dashed lines show the histogram obtained for 103-nm-diameter polystyrene latex spheres added subsequently to the virus. The sample consisted of 0.40 ml of the preparation of viruses mixed with 4 ml of RPM1 1640 nutrient medium containing 8% fetal calf serum and 0.06 ml of Tween 80. The mixture was filtered with 0.2~pm Nuclepore. The suspension of viruses flowed through a 3IPnm-diameter pore, 2.58 pm long. for 297 s at 9 in of water pressure. with 1.000 V across the pore and 38.0 nA passing through it.
284
DEBLOIS
ET AL.
axial ratios and thus different friction factors, and the light scattering is not linear with particle size, but varies as approximately r4 in this size range. For the Tipula iridescent virus, the measured hydrodynamic diameters are given without further corrections because, for comparison, the icosahedral dimensions of the virus, i.e., face-to-face and apex-to-apex distances, were not clearly discerned in the electron micrographs and only some average spherical diameter was obtained. The results clearly indicate that the hydrodynamic size is much larger than the virus size determined either by electron microscopy or by resistive-pulse analysis. We had taken precautions to eliminate debris and virus aggregates from the samples by filtering and by adding surfactant. One explanation for the observed discrepancy is that there may be a peripheral fuzzy coat surrounding the TIV, as reported by Stoltz (22). Such an amorphous outer layer would have the most effect on hydrodynamic properties of the virus and would have relatively little effect on the resistive-pulse determination because the conductivity of the fuzzy region would not differ very much from the conductivity of the surrounding fluid. The fuzz was not visible in our micrographs, but it is apparently of such low density that it is not visible using ordinary techniques. Near the end of this investigation a photon-counting system was completed. The results obtained with this system verified the earlier measurements on the polystyrene spheres as well as the virus samples. Figure 5 is an example of the light-scattering data showing the 100 channels of output on a semilog plot for gypsy moth virus. Above the data points is shown an autocorrelation function that was generated numericahy by using the multiplet distribution determined in Fig. 3, the appropriate diffusion coefficient for each component, and a lightscattering weighting factor of r4.3 as an approximation to the departure from true Rayleigh scattering for particles of this size (15). The numerical simulation of the correlation function based on the resistive-pulse data gives a straight line for the logarithm of the autocorrelation function. The slope of this line is close to the experimentally observed slope. Furthermore, the curvature in the numerically generated function is far less than the scatter in the experimental data. Thus, the further analysis of light-scattering data by expansions into cumulants was not useful in these cases. Other workers studying viruses have also come to this conclusion regarding cumulant analysis with limited signal to noise conditions (15). Electron Microscopy
Figure 6 shows typical micrographs of the three types of viruses studied. The rod viruses appear as right circular cylinders with hemispherical
INSECT VIRUS SIZE AND POLYDISPERSITY
285
GMV
FIG. 5. The autocorrelation function of laser light scattered at 90” (homodyne) from gypsy moth virus in water at room temperature. Tau is the delay time between successive samplings of the light intensity. The dots in the lower line are the experimental data. The solid line connects points numerically calculated from the size distribution determined in Fig. 3. A weighting factor of r4.3 was used to approximate the variation in optical cross section as a function of size. Diffusion constants for each multiplet component corresponded to the volume and the friction factor of that component. The upper curve is displaced to the right by 0.2 ms units for clarity.
end caps (23). For a cylindrical radius b and total length of 2a, the volume of such particles is V = 2&+‘a(l-b/3a). The results of the volume measurements from the electron micrographs are indicated in Table 1. Histograms of axial ratios were also established and these were used to correct for shape in determining equivalent spherical diameters both for the resistive-pulse measurements and for the light-scattering measurements. The number of enveloped GMV rods counted was only 16 and for this reason no multiplet structure could be observed in particle number vs. volume. In the European pine sawfly virus preparations, we counted 20 spherical globules among the virus rods. These are presumed to be curled-up enveloped rods (6,7).
286
DEBLOIS
ET AL.
INSECT
VIRUS
SIZE
AND
POLYDISPERSITY
287
Tipuln iridescent viruses were photographed at 300,000x magnification and were basically round in cross section with hints of flat-sidedness as evidence of their icosahedral shape (Fig. 6). A histogram of 21 viruses yielded a sharp peak representing a volume of (2.6 2 0. I) x IO-l5 cm3 from the average measured diameter of 170 + 2 nm. A much smaller peak representing only seven viruses indicated a possible secondary group with a diameter of 162 & 4 nm. The results in Table 1 indicate substantial agreement between the measurements of size by resistive-pulse, light-scattering, and electron microscope techniques. The first two methods measure hydrated virus size while the last measures the dehydrated virus size. The data of Table 1 show no significant shrinkage in size due to dehydration for the GMV and EPSV unencapsulated rods. For the encapsulated rods of GMV the electron microscope measurements were too few in number to resolve the multiplets; thus an accurate comparison with the resistive-pulse values could not be made. Dehydration may produce a small shrinkage in Tip& iridescent virus but does not appear to affect the encapsulated EPSV rods. Separate components of the polydisperse virus preparations could not be resolved by laser light scattering and these measurements were heavily biased toward the largest components present in a sample, as expected from Mie scattering theory (15). The light-scattering measurements for Tipulu iridescent virus show the largest departure from the other two methods, possibly because of a fuzzy, amorphous outer coat. The electron microscopy and resistive-pulse sizes for TIV are in basic agreement if one takes into account the spread in sizes visible in the electron micrographs. An important result of this investigation is that the Nanopar analyzer, using the resistive-pulse technique, is able to resolve distinctly the various components of a polydisperse virus sample and to measure their absolute volumes when the shapes are known. Data are gathered with greater ease than by electron microscopy and with probably greater accuracy, since preparation for electron microscopy may lead to particle distortion. Thus, the analyzer may be especially suitable for characterizing or classifying polydisperse viruses. Virus sizes in a given run are measurable to several nanometers or better. The smallest virus sized thus far is 59 nm in diameter (24). Signalto-noise observations indicate that viruses of half that diameter could be sized under special conditions of cleanliness, high electrolyte concentration, and small pore diameter. Our measurements by resistive-pulse analysis and electron microscopy agree with previous observations of two shapes of virus particles, rodshaped and globular, in some preparations of the European pine sawfly virus, and of varying numbers of virus rods enveloped in single outer membranes for the gypsy moth virus. In addition, these techniques may
DEBLOIS
288
have sized out a structural virus.
ET At.
variant in a preparation
of Tipula iridescent
ACKNOWLEDGMENTS We thank R. R. Russell for the electron microscopy and M. V. Doyle for analytical help.
REFERENCES 1. Carlson, F. D. (1975) Annu. Rev. Biophys. Bioeng. 4, 243-264. 2. Cummins, H. Z., and Swinney, H. L. (1970) in Progress in Optics (Wolf, E.. ed.), Vol. 8, pp. 135-200, North-Holland, Amsterdam. 3. DeBlois, R. W., and Bean, C. P. (1970) Rev. Sci. lnstrum. 41, 909-916. 4. DeBlois, R. W., and Wesley, R. K. A. (1977) .I. Viral. 23, 227-233. 5. DeBlois, R. W., Bean, C. P., and Wesley, R. K. A. (1977) J. Collvid Interface Sci. 61, 323-335. 6. Bahr. G. F., Engler, W. F., and Mazzone. H. M. (1976) Quart. Rev. Biophys. 9, 459-489. 7. Mazzone, H. M., Breillatt, J. P., and Anderson, N. G. (1970) in Proceedings of the IVth International Colloquium on Insect Pathology, pp. 371-379, College Park, Maryland. 8. Bergold, G. H. (1953) in Advances in Virus Research. (Smith, K. M., and Lauffer. M. A., eds.) Vol. 1, pp. 91-139, Academic Press, New York. 9. Bergold, G. H. (1963) in Insect Pathology (Steinhaus, E. A., ed.). Vol. 1 pp. 413456. Academic Press, New York. 10. Harrap, K. A. (1972) Virology 50, 133-139. 11. Mazzone, H. M., Breillatt, J. P., and Bahr, G. F. (1973) in Fifth International Colloquium on Insect Pathology and Microbial Control, p. 42, Oxford University. 12. Osbom, J. A. (1945) Phys. Rev. 67, 351-357. 13. Stoner, E. C. (1945)Phil. Mag. 36, 803-821. 14. Pusey, P. N., Koppel, D. E., Schaefer, D. W., Camerini-Otero, R. D.. and Koenig. S. H. (1974) Biochemistry 13, 952-960. 15. Salmeen, I., Rimai, L., Luftig, R. B., Liebes, L., Retzel, E., Rich, M., and McCormick, J. J. (1976) J. Virol. 17, 584-596. 16. Bargeron, C. B. (1974) J. Chem. Phys. 60, 2516-2519. 17. Koppel, D. E. (1972)J. Chem. Phys. 57, 4814-4820. 18. Wrigley, N. G. (1968) J. Utrastruct. Res. 24, 589-590. 19. Breillatt, J. P., Brantley. J. N., Mazzone. H. M., Martignoni, M. E., Franklin, J. E., and Anderson, N. G. (1972) Appl. Microbial. 23, 923-930. 20. Pecora, R. (1965) J. Chem. Phys. 49, 1036-1043. 21. Tanford, C. (1961) Physic& Chemistry of Macromolecules, pp. 324-328, Wiley, New York. 22. Stoltz, D. B. (1971) J. Ultrastruct. Res. 37, 219-239. 23. Mazzone, H. M., and Tignov, G. H. (1976) in Advances in Virus Research, Vol. 20, pp. 237-270, Academic Press. New York. 24. Feuer, B., Uzgiris, E. E., DeBlois, R. W.. Cluxton, D. H., and Lenard, J. (1978) Biophys. J. 21, 38a.
