Analysis of a plaque assay method for purified poliovirus MEF-1

VIROLOGY 13, 427--438

(1961)

Analysis of a Plaque Assay Method for Purified Poliovirus MEF-1~ J O Y C E T A Y L O R AND A. F. G R A H A M

Connaught Medical Research Laboratories, University o] Toronto, Canada, and The Wistar Institute o] Anatomy and Biology, Philadelphia, Pennsylvania Accepted December 20, 1960 Purified poliovirus had a particle to PFU ratio of 48 and, after correction for inefficient adsorption in the assay, this ratio was reduced to 30. All the assay cells were infectible and the number of cells infected corresponded to the number killed. The ratio of 30 could be explained by assuming that each cell had 29 abortive adsorption sites for every entry site, or that the virus population was heterogeneous with 29 defective particles for every particle capable of infecting, or by a combination of these principles. INTRODUCTION

kidney cells were used (Dulbecco and Vogt, Present methods of purifying poliovirus 1954) and, unless otherwise specified, were yield a product t h a t is uniform with respect cultivated in a medium consisting of Earle's to particle size but t h a t contains a large ex- saline solution (in which the bicarbonate cess of particles t h a t are apparently non- concentration was reduced to 0.55 g per infectious. Thus, for the M E F - 1 strain, liter), 0.5 % lactalbumin hydrolyzate, and Schwerdt and Fogh (1957) determined a 5 % ox serum. The p H of the medium was physical particle to infectious particle ratio maintained at 7.4 b y a 0.01 M concentration of 60. Our estimates for the same virus puri- of Tris buffer, and the use of COe atmosfied in a quite different way gave similar phere during cellular multiplication was unnecessary. results, as reported here. Preparation o/ cell suspensions / t o m I n the present p a p e r we have a t t e m p t e d to monolayers. Suspensions of single cells analyze some of the factors t h a t might conwere obtained from monolayers b y the foltribute to the disparate ratio. Preliminary discussions of some of the data have al- lowing t r e a t m e n t : The cell layer on a 10-cm ready been presented ( T a y l o r and G r a h a m , petri dish was washed twice with PBS (phosphate-buffered saline, Dulbecco and 1959; G r a h a m , 1959). Vogt, 1954) lacking calcium and magnesium, and then incubated for 5 minutes MATERIALS AND METHODS with a solution of 0.25 % trypsin and 0.05 % Virus. The M E F - 1 strain of poliomyelitis Versene in calcium- and magnesium-free virus was used throughout. The procedure PBS. I f necessary the cells were gently for labeling this virus with p32, and for its pipetted once or twice and an equal volume purification, has been described (Taylor of medium containing 5 % ox serum was and G r a h a m , 1958). added to stop the action of Versene and Cells. P r i m a r y cultures of rhesus m o n k e y trypsin. T h e cells were then r e a d y for ex1This work was supported in part by grants perimental use. T h e y were not centrifuged. Virus assay. M o n k e y kidney cells were from the National Institutes of Health of the United States Public Health Service (E-262), the cultivated in 10-cm petri dishes until a National Foundation, the National Cancer Insti- complete sheet was formed. T h e cell layers tute of Canada, and the W. B. Boyd Memorial were washed once with PBS and 0.5 ml Fund. of the virus suspension in PBS was applied 427

428

JOYCE TAYLOR AND A. F. GRAHAM

to each plate. An adsorption period of 90 minutes at 37 ° was allowed, and 12 ml of agar overlay was then poured on each plate. The overlay consisted of Earle's saline (2.2 g per liter of bicarbonate) containing 1 % agar, 0.1% yeast extract, and 0.1% bovine or human albumin. The plates were maintained in a humidified 5 % COo atmosphere for 4 days, the cell layers were stained by addition of 1/20,000 neutral red in PBS and the plaques scored. Four plates, between 20 and 50 plaques per plate, were employed for each assay. Titration o] infected cells. Virus was added to a monolayer of cells. After an adsorption period of less than 2 hours, the cells were washed five times with PBS, suspended as described in a preceding section, enumerated in a counting chamber, and diluted in PBS. Half-milliliter samples from the diluted suspension were placed on monkey kidney cell monolayers that had previously been washed with PBS. Immediately afterward, 2 nfl of melted overlay was added to the plates. When this agar had solidified a further 10 ml of overlay was added and the plates were incubated for 4 days at 37 ° in a 5 % COo atmosphere. The cell layers were then stained by addition of 1/20,000 neutral red solution and the plaques were scored. Generally, a set of four plates was utilized for each titration. There was a linear relationship between the number of cells plated and the number of plaques formed. Free virus did not contribute to the formation of plaques since 99 % of the plaque-forming units (PFU) in the infected cell suspension sedimented with the cells. Cloning eJficiencg of cells. When monkey kidney cells were plated according to the technique of Puck et al. (1956) a fraction of the population gave rise to macroscopic colonies; this fraction will be designated the cloning efficiency. All cells that were to be used in cloning experiments were propagated in a medium consisting of Hanks' salt solution supplemented with 0.5 % of laetalbumin hydrolyzatc and 1 % of ox serum. The p H was maintained at 7.4 with bicarbonate, the cells being grown in stoppered bottles. To determine the cloning efficiency of these cells, the layers were

washed five times with PBS, suspended, counted, and diluted in medium. The medimn consisted of solution CMRL-1066 (Parker et al., 1957) supplemented with 20 % ox serum. One-tenth-milliliter samples of this dilution were added to each of six 60-mm petri dishes containing 5 ml of the same medium. After i0 days at 37 ° in a humidified atmosphere of 5 % CO2 in air, the colonies were fixed, stained with Giemsa, and scored. There was a linear relationship between the number of cells plated and the number of colonies formed. The cloning efficiency varied from i0 % to 25 % in different experiments. In cloning infected populations, the spread of virus from infected cells was prevented by the use of an ox serum that was virueidal in the cloning medium (Takemori et al., 1958). At the concentration used (20 % ox serum) the addition of 5 × 10 ~ P F U of poliovirus to each plate did not reduce the cloning efficiency of uninfected cells. EXPERIMENTAL

Ratio of Physical to Infectious Units in Purified Virus Virus, adsorbed on a Dowex-1 resin colunm, was eluted with 0 . 0 5 M NaC1 in 2-ml fractions (Taylor and Graham, 1958), each fraction being collected in 2 ml of twofold concentrated PBS. The fractions containing virus were combined and stored in the frozen state until required for assay. Samples were removed from the thawed specimen for assay by the standard plaque procedure at the same time as they were taken for determination of particle concentration. The concentration of physical partieles was estimated by a technique developed for small concentrations of poliovirus (Pinterie and Taylor, in preparation). Briefly, drops of virus suspension to which had been added a known number of latex reference particles were applied to a Formvar film floating on the surface of an ammonium acetate-ammonium carbonate buffer. When dialysis against the buffer was complete, the film supporting the drop was lowered onto a gold grid, and the drop was allowed to evaporate under carefully

PLAQUE ASSAY FOR POLIOVIRUS

429

FIG. 1. Purified preparation of poliovirus MEF-1 as seen in the electron microscope. The large spheres are latex, 88 m~ in diameter, Magnification: × 50,000. controlled conditions. The specimens were shadow-cast with palladimn and covered with a carbon film. A typical field is shown in Fig. 1. Values of the ratio of physical to infectious units are listed in Table 1 for four preparations of purified virus. The average ratio is 48, in good agreement with the value given by Schwerdt and Fogh (1957) for partially purified M E F - 1 virus assayed on hmnan amnion cells. A high total particle to infectious particle

ratio would be obtained if (1) conditions of assay were unfavorable for attachment of virus to the cell monolayer, either during the adsorption period or from the agar overlay; (2) only a fraction of adsorbed infectious particles go on to multiply; (3) the virus population were heterogeneous with respect to capacity either to adsorb, or, once adsorbed, to initiate the formation of viral progeny. E~ch of these possibilities will be considered.

430

J O Y C E T A Y L O R A N D A. F. G R A H A M TABLE 1

RATIO OF TOTAL TO INFECTIOUS PARTICLES IN PURIFIED SUSPENSIONS OF POLIOVIRUS MEF-1

Preparation PFU per mD number 1 2 3 4

3.8 1.6 3.0 6.3

X X X X

Total particles per ml

Ratio total particles per ml PFU per ml

107 107 107 108

1.59 X 109 3.8 X 10s 3.0 X 109 1.78 X 101° Average ratio

42 24 100 28 48

P F U = plaque-forming units. IO0 bJ m

so r,o z

60 n

o

n*

N , ,

I

,

,

,

I

I

,

2

,

I

3

HOURS

FIG. 2. The rate of adsorptioR of infectivity and p~a to monkey kidney cell monolayers from a purified, P*Mabeled preparation of MEF-1 virus. Crosses represent virus; open circles, ps=.

Adsorption o] Virus to Monlcey Kidney Cell Monolayers Figure 2 illustrates the results of an experiment designed to measure the rates of adsorption of pa2 and infectivity to monkey kidney cell monolayers from a purified, labeled, virus preparation. To each of a series of monkey kidney cell layers in 10-cm petri dishes was added 0.1 ml of PBS containing 107 P F U of Pa2-1abeled virus. The plates were placed at 37 °. At intervals two plates were removed and 4.9 ml of PBS was added to each. After thorough mixing, samples of each supernatant fluid were assayed for unadsorbed pa2 and virus. While the over-all rates of adsorption of pa2 and virus, described by the broken line in Fig.

2, are exponential, there is considerable scatter of the experimental points as might be expected from this type of experiment. Nevertheless, there is good correspondence between the amounts of pa2 and virus infectivity adsorbed for most of the sets of monolayers. This experimental result is in agreement with one published earlier (Taylor and Graham, 1959). It is concluded that the major portion of the labeled particles was homogeneous with regard to adsorption rate, and that the pa2 marker could be used as a measure of adsorption of infectious units. In the experiment just described, conditions were necessarily chosen so that the major portion of the virus would be adsorbed. Such conditions could not be used in the plaque assay procedure since the small amount of fluid would not readily permit uniform distribution of virus over the plate. When a larger volume of virus containing fluid is used over the monolayer to improve the distribution, the rate of virus adsorption is markedly decreased. The rate of adsorption-fluid volume relationship for MEF-1 virus is shown in Fig. 3, and is similar to that described by Bachrach et al. (1957) for foot-and-mouth disease virus. To determine the amount of virus that adsorbed directly to the cell monolayers

under the standard conditions of plaque assay the following experiment was done. Monolayers of monkey kidney cells were washed with PBS, and to each layer was added 0.5 ml of a purified, labeled, virus suspension containing about 2 × 107 PFU per milliliter. After incubation for 90 rainutes at 37 °, the cell layers were washed free of unadsorbed virus with PBS, and the cells were suspended and assayed for pa2 content. The fraction of pa', that remained adhering to the cells was assumed to represent the amount of infectious virus firmly adsorbed. On the average, about 17% of the pa2 was adsorbed, as the results of Table 2 show. As an independent check on this estimate of adsorbed virus, from Fig. 2 it may be determined that about 40 % of virus would be adsorbed in 90 minutes from a volume of 0.1 ml. Correction of this value by the factor 0.55, derived from Fig. 3 for an adsorption volmne of 0.5 ml,

PLAQUE ASSAY FOR POLIOVIRUS i n d i c a t e s t h a t a b o u t 22% of t h e P F U s h o u l d h a v e been a d s o r b e d , in a g r e e m e n t w i t h t h e p32 d a t a .

Adsorption of Virus to Monolayers ]rom the Agar Overlay In the standard assay procedure, virus remaining unadsorbed after the preliminary a d s o r p t i o n p e r i o d is n o t r e m o v e d b u t becomes m o r e or less u n i f o r m l y s u s p e n d e d in a g a r w h e n t h e o v e r l a y is poured. P a r t of this v i r u s l a t e r diffuses t h r o u g h t h e o v e r l a y a n d gives rise to p l a q u e s ( Y o u n g n e r , 1956). T h e following e x p e r i m e n t w a s done to d e t e r m i n e w h e t h e r t h e a d s o r p t i o n of v i r u s f r o m t h e a g a r o v e r l a y w a s complete. A 1-ml s a m p l e of a d i l u t e v i r u s s u s p e n sion (the e x a c t c o n c e n t r a t i o n of v i r u s is u n i m p o r t a n t ) w a s a d d e d to each of a n u m b e r of w a s h e d m o n k e y k i d n e y cell m o n o l a y e r s w h i c h were t h e n i n c u b a t e d a t 37 °. A t i n t e r v a l s , 12 ml of a g a r o v e r l a y w a s a d d e d to each of a set of t h r e e p l a t e s . A f t e r f u r t h e r I00

.5

~E "7 0 o z

80

~:

60

l,g I-I'll,.

o

0

40

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3,

4

5

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FIG. 3. The adsorption of poliovirus to monkey kidney cell monolayers from different volumes of phosphate-buffered saline. A purified suspension of MEF-1 virus was diluted in PBS and various volumes were added to monkey kidney cell monolayers. Four plates were used for each volume. After 2 hours' adsorption at 37°, unadsorbed virus was removed, the cell layers were washed twice with PBS, and overlays poured. Plaques were scored after 4 days, and the average number of plaques for each set was converted into a percentage of the number of plaques found with an adsorption volume of 0.1 ml.

431 TABLE 2

THE

ADSORPTION OF p3~ TO M O N K E Y K I D N E Y C E L L S FROM A P U R I F I E D SUSPENSION OF P32-LABELED POLIOVIRUS a

Virus prepara-

tion 1 2 3

Batch no. of cells

p32 added

p32 ad-

to cells sorbed to % ps2 ad(cpm) b cells (cpm) sorbed

(i) (ii)

207 207

39 21

19 10

(i) (ii)

501 501

75 83

15 17

(i) (ii)

734 734

134 154

18 21

Virus was adsorbed from 0.5 ml of PBS to a monolayer of monkey kidney cells in a 10-era dish for 90 minutes at 37 ° C. Each estimate of adsorption is the average for two monolayers from a given batch of cells. b cpm = Counts per minute. i n c u b a t i o n for 4 d a y s , t h e m o n o l a y e r s were s t a i n e d w i t h n e u t r a l r e d a n d t h e n u m b e r of p l a q u e s scored. T h e results, shown in Fig. 4, i n d i c a t e t h a t t h e t i t e r of t h e o r i g i n a l v i r u s s u s p e n s i o n i n c r e a s e d m a r k e d l y as t h e preliminary adsorption period was lengthened to 3 hours. H e n c e t h e diffusion of v i r u s t h r o u g h t h e a g a r o v e r l a y is f a r f r o m c o m p l e t e u n d e r s t a n d a r d a s s a y conditions. T h e d a t a of Fig. 4 a l l o w a f u r t h e r estim a t e to be m a d e of tile a m o u n t of v i r u s a d s o r b e d to a m o n o l a y e r in 90 m i n u t e s , t h a t is, d u r i n g a t i m e e q u i v a l e n t to t h e p r e l i m i n a r y a d s o r p t i o n p e r i o d of t h e s t a n d a r d p l a q u e a s s a y . I t m a y be seen t h a t a t l e a s t 90 P F U ' s were a d d e d to each p l a t e . T h e c u r v e of Fig. 6 r e p r e s e n t s a c o m p l e x s i t u a tion, b u t a b o u t 20 P F U ' s , or n o t m o r e t h a n 22 % of those a d d e d , a d s o r b e d in 90 m i n utes. T h e r e m a i n d e r of t h e 70 p l a q u e s o b s e r v e d a t 90 m i n u t e s r e p r e s e n t e d v i r u s t h a t h a d diffused t h r o u g h t h e o v e r l a y . 2 I f t h e -~In fact, as will be shown in the next section, the plating efficiency of virus in the routine plaque assay is 60 %. From this value, and the total number of plaques obtained at 90 minutes (Fig. 6), it is calculated that 120 PFU were added to each plate. Hence the fraction of this amount that adsorbed directly to the monolayer in 90 minutes was 20/120, or 17 %, an estimate consistent with

432

JOYCE TAYLOR AND A. F. GRAHAM 9O

so

>

~7o 5

°

~' 6 o z

5(3

I 0

I ADSORPTION

I 2 TIME

I 3 (HOURS)

FIG. 4. E f f e c t of a d s o r p t i o n t i m e p r i o r to a d d i t i o n of overlay on the number of plaques

formed on monkey kidney cell monolayers. Unadsorbed virus was not removed before addition of overlay. results of Fig. 4 are plotted on a logarithmic ordinate the curve takes the same shape as that described by Youngner (1956) for M E F - 1 virus.

Correction. for Incomplete Adsorption in Virus Assay The results presented in the preceding two sections together indicate that the conditions of assay did not permit direct titration of all the virus in a suspension. Clearly, the error was not much greater than a factor of 2, but since a more accurate estimate was desired, the following experiment was carried out: Three sets of monkey kidney monolayers were prepared in 10-cm petri dishes. To each plate of the first set were added 50-100 infectious particles in 0.5 ml of PBS; the exact number of infectious units was unknown at this stage and will be designated N. After a 90-minute adsorption period, the supernatant liquid from each plate was transferred to a plate of the second set. The cell layers of the first set were each washed with 0.5 ml of PBS and the washings were transferred to the plates of the third set. that obtained from P~ adsorption data described in the preceding section.

The plates of the first set were then overlaid with agar. After 90 minutes for adsorption, the supernatant liquids from the second and third sets of plates were discarded, the cell layers washed, and overlays poured. Three days later, the plaques on a l l three sets were counted. If x, y, and z, are the average numbers of plaques on the first, second, and third sets of plates, respectively, then x / N = (y + z ) / ( N -- x) on the assumption t h a t the rate of adsorption of virus to the cell layers is independent of virus concentration. From this expression, N, the number of P F U in the original suspension, can be evaluated. The results of several such experiments are shown in Table 3. Essentially, the above experiment estimates the correction for inefficient adsorption of virus in the standard assay procedure. N is the maximum number of infectious units in a given suspension that can initiate plaques under our particular conditions o/ assay (Taylor and Graham, 1959). As will be seen from Table 3 the routine technique estimates, on the average, about 60 % of N, and this fraction will be designated as the plating efficiency. When corrected for plating efficiency, the average total particle to infectious particle ratio is reduced to about 30, a relatively insignificant change.

Susceptibility o/ Monkey Kidney Cells to Virus The N values of Table 3 would represent the true number of infectious units in a virus suspension if all the particles capable of intracellular multiplication were to form plaques, and we now have to inquire what factors might contribute to a reduced value of N. An obvious reason for failure of an infectious particle to form a plaque would be its adsorption to a cell that, under any circumstances, could not support its multiplication. Whether such a heterogeneity of cells exists in the primary monkey kidney populations was determined by the following experiment: M o n k e y kidney cells were propagated in 10-cm dishes until approximately twothirds of the area of each plate was covered with cells. The layers were washed with PBS, and 2 ml of a suspension of purified M E F - 1 virus in PBS was added to each:

PLAQUE ASSAY FOR POLIOVIRUS

433

TABLE 3 ESTIMATION OF THE N U M B E R OF INFECTIOUS U N I T S IN SUSPENSIONS OF P O L I O

Virus preparation

3

Number of plates per plate in series

MEF-1 a

x

y

z

Calculated number of infectious units (N) added to series xb

23.5 39.5

12.6 11.5

3.8 3.0

78 63

1.6 X 109 1.3 X 109

8.3 X l0 s 7.6 X 108

52 58

24.8 28.0

15.8 10.0

2.8 2.3

98 50

9.8 X l0 s 5.0 X l0 s

5.6 X l0 s 3.2 X l0 s

57 64

20.8

6.8

1.8

35

7.0 X 10s

5.9 X l0 s

84

Calculated titer of virus preparation (PFU per ml)

Titer of virus preparation by Plating standard pro- efficiency of cedure (PFU standard per ml) ' procedure

Table reproduced from Taylor and Graham (1959). b Calculated from the expression

x N t h e m u l t i p l i c i t y of i n f e c t i o n w a s m u c h g r e a t e r t h a n 1. A f t e r i n c u b a t i o n a t 37 ° for 90 m i n u t e s , u n a d s o r b e d v i r u s w a s r e m o v e d b y w a s h i n g t h e l a y e r s five t i m e s w i t h P B S a n d a s s a y e d . T h e cells were s u s p e n d e d , counted, a n d p l a t e d for i n f e c t i o u s centers. I n some e x p e r i m e n t s p a r t of tim s u s p e n s i o n of i n f e c t e d cells w a s r a p i d l y frozen a n d t h a w e d s e v e r a l t i m e s a n d a s s a y e d for virus. T h e r e s u l t s of s e v e r a l e x p e r i m e n t s a r e shown in T a b l e 4, a n d i t is c l e a r t h a t t h e efficiency of p l a t i n g i n f e c t e d cells w a s close to 100 %. A n e g l i g i b l e n u m b e r of P F U rem a i n e d a f t e r freezing a n d t h a w i n g t h e inf e c t e d cells, i n d i c a t i n g t h a t m o s t of t h e v i r u s a d s o r b e d to t h e cells h a d e n t e r e d t h e eclipse p h a s e a n d w o u l d n o t be r e l e a s e d as free v i r u s d u r i n g a s s a y of i n f e c t i o u s centers. I t is p r o b a b l y t h e r e f o r e , t h a t m o s t of t h e cells of t h e p r i m a r y m o n k e y k i d n e y p o p u l a t i o n s c a n s u p p o r t m u l t i p l i c a t i o n of virus, as h a s a l r e a d y b e e n i n d i c a t e d b y D u n n e b a c k e a n d R e a u m e (1958) for t h e M a h o n e y s t r a i n of p o l i o v i r u s . As a p o i n t of t e c h n i q u e i t s h o u l d be m e m t i o n e d t h a t t h e f r a c t i o n of cells in a l a y e r t h a t b e c o m e i n f e c t e d m a y fall off m a r k e d l y if t h e glass s u r f a c e is c o m p l e t e l y covered w i t h cells b e f o r e a d d i t i o n of virus. E i t h e r some cells do n o t g e t e x p o s e d to v i r u s in t h e c o m p l e t e l a y e r or, p e r h a p s , m a n y cells a r e in t h e s t a t i o n a r y p h a s e of t h e g r o w t h cycle

y+z N-x" TABLE 4 TITRATION OF MONKEY KIDNEY CELLS INFECTED WITH HIGH CONCENTRATIONS OF POLIOVIRUS MEF-1 Multiplic- Number of Expt. ity" of cells exposed no. infection to virus

Number of % Cells cells giving giving plaques plaques

1

100:1 100:1

1.5 X 105 1.7 X 105

1.0 X 10 '~ 1.6 X 10 5

67 94

2

20:1 20:1

1.8 X 106 2.1 X 106

1.8 X 106 2.0 X 106

100 95

5:1 5:1 5:1

2.3 x 105 3.8 x 10 '~ 3.2 N 10 ~

2.5 x 10 .~' 2.7 x 105 2.9 X 105

109 71 91

Determined from the ratio PFU adsorbed cells exposed to virus. The nmnber of PFU adsorbed was determined by following disappearance of virus fronl the supernatant fluid over the monolayer. a n d n o t suiticienly r o b u s t to w i t h s t a n d t h e subsequent manipulation.

Relation between Multiplicity of Infection and Number of Cells Infected U n d e r c o n d i t i o n s of p l a q u e a s s a y t h e m u l t i p l i c i t y of i n f e c t i o n is v e r y low. I t w a s n e c e s s a r y , t h e r e f o r e , to d e t e r m i n e t h e r e l a tionship between multiplicity and fraction of cells i n f e c t e d for t h e m o n k e y k i d n e y cell

434

JOYCE TAYLOR AND A. F. GRAHAM

° FII

/11

0.4

0-2

0~i

~"

0

i

I

@5 1.0 M U L T I P L I C I T Y (m)

I

I-5

Fro. 5. Theoretical curves and experimental data showing the relationship between multiplicity of infection, M, and fraction of cells infected, P, in monkey kidney cell populations infected with MEF-1 virus. Curve A, P = 1 -- e". The derivation of curves B, C, D, E, and F is explained in the text. populations. Assuming a particle to P F U ratio of 30, there are several hypothetical situations t h a t m a y be considered beforehand. Firstly, all the cells m a y support virus multiplication after adsorption of one infectious unit, so t h a t all particles infectious for m o n k e y kidney cells are estimated in the plaque assay. This situation would exist if the viral population were heterogeneous, with only one of every 30 particles capable of infecting a cell. T h e relationship between multiplicity of infection, m, and fraction of cells infected, P, should be given by the first t e r m of the Poisson distribution, P = 1 - e -'~. Secondly, there are three general situations in regard to cell susceptibility t h a t could contribute to reduced titer of inlettious virus : (1) An adsorbed virus could fail to multiply if the cell surface were heterogeneous, even if the cell population and the viral population were homogeneous. Thus, 1 in 30 adsorbed particles would multiply if only

1/30th of the adsorption sites would permit multiplication of the virus; for descriptive purposes an adsorption site t h a t permits infeetion will be designated an entry site. The relationship between fraction of cells infected and multiplicity of infection would then be P = 1 - e -~/a°, where x is the number of particles adsorbed per cell, or P = 1 - e-'% where m is the n u m b e r of P F U adsorbed per cell. This ease is, therefore, formally indistinguishable from the case of heterogeneous virus population. (2) Assume t h a t the cell population is heterogeneous and every particle of the viral population is infectious. A particle to P F U ratio of 30 would be observed at low multiplicity of infection if only 1 in 30 cells could be infected with a single particle. At higher nmltiplieities the P to z relationship would begin to deviate from the simple Poisson fornmla since the fraction of cells t h a t adsorb more t h a n 1 particle would increase. The hypothetical ease to consider is t h a t in which 1 cell in 30 could be infected by a single particle, and the remaining 29 cells would require more than one particle. Then the fraction of cells infected at a specific multiplicity, x, would be p =

fraction of cells adsorbing one particle 30 -4- fraction of cells adsorbing n or more particles

These terms m a y be calculated from the Poisson formula for different values of x (particle multiplicity) and n. I n Fig. 5, P has been plotted against m (nmltiplicity of P F U ) , where m = x/30, for values of n = 2, 5, 10, 15, and 20 (curves B, C, D, E, and F, respectively). I t will be noted, for example, t h a t for n = 2, 100 % of the cells should be infected at m = 0.4 since x = 12 at this point and most cells have adsorbed more than 2 particles. This method of expression facilitates comparison with the experimental results which are obtained in terms of PFU. The curves are different from t h a t for P = 1 -- e -'~ superimposed in Fig. 5 as curve A. (3) Each cell might be infected b y one adsorbed particle, provided it adsorbed in

PLAQUE ASSAY FOR POLIOVII~US the r i g h t place, b u t t h e r a t i o of a d s o r p t i o n sites to e n t r y sites could v a r y f r o m cell t o cell. T h i s t y p e of h o s t h e t e r o g e n e i t y h a s been d e s c r i b e d ( R e i d a n d M a e L e o d , 1954). A p p l i e d to t h e p r e s e n t case t h e r e l a t i o n s h i p b e t w e e n P a n d m w o u l d follow a n e g a t i v e b i n o m i a l d i s t r i b u t i o n differing f r o m t h e s i m ple P o i s s o n expression for a n y s i g n i f i c a n t degree of h e t e r o g e n e i t y . I t is difficult to p l o t t h e o r e t i c a l curves for this case, b u t if i t is f o u n d t h a t t h e e x p e r i m e n t a l d a t a fit t h e d i s t r i b u t i o n P = 1 - e - ~ t h e y c a n n o t fit the negative binomial distribution and this t h i r d p o s s i b i l i t y could b e r e j e c t e d . To determine experimentally the fraction of cells i n f e c t e d a t different m u l t i p l i c i t i e s of a d s o r b e d virus, i n c o m p l e t e l y f o r m e d l a y e r s of cells in 10-cm dishes were w a s h e d w i t h P B S a n d t h e n exposed to 5 - m l v o l u m e s of a s u s p e n s i o n of p u r i f i e d P3-%labeled virus. The virus was adsorbed from relatively l a r g e v o l u m e s to i n c o m p l e t e l a y e r s to i n s u r e t h a t t h e cells w o u l d be u n i f o r m l y e x p o s e d to virus. T h e m u l t i p l i c i t y of i n f e c t i o n w a s a l t e r e d b y a d d i n g different c o n c e n t r a t i o n s of

435

v i r u s or b y v a r y i n g t h e l e n g t h of a d s o r p t i o n period. U n a d s o r b e d v i r u s w a s r e m o v e d b y w a s h i n g five t i m e s w i t h P B S , t h e cells were s u s p e n d e d , counted, a n d p l a t e d on fresh m o n o l a y e r s for i n f e c t i o u s centers. T o d e t e r m i n e t h e n u m b e r of i n f e c t i o u s u n i t s a d s o r b e d to t h e cells, N w a s first d e t e r m i n e d for each v i r u s p r e p a r a t i o n as d e s c r i b e d in a p r e v i o u s section. T h e f r a c t i o n of v i r u s a d s o r b e d w a s t a k e n as e q u a l to t h e f r a c t i o n of p3.., a d s o r b e d b y t h e cells, a n d t h e a m o u n t of p3-- a d s o r b e d w a s d e t e r m i n e d b y d i r e c t e s t i m a t e on t h e i n f e c t e d , s u s p e n d e d , cells. T h e a m o u n t of p3.- a d s o r b e d could be d e t e r m i n e d o n l y w h e n t h e m u l t i p l i c i t i e s of infection were g r e a t e r t h a n 0.2, a n d p r o b a b l y t h e e s t i m a t i o n s were n o t v e r y a c c u r a t e even a t t h e h i g h e r m u l t i p l i c i t i e s owing to t h e r e l a t i v e l y s m a l l a m o u n t of v i r u s a d s o r b e d . T a b l e 5 shows t h e r e s u l t s of t h r e e e x p e r i ments, each performed with a different virus p r e p a r a t i o n . I n e x p e r i m e n t 3, it w a s a s s u m e d t h a t t h e s a m e f r a c t i o n of virus, 0.045, a d s o r b e d a t each d i l u t i o n a l t h o u g h the adsorption was measured directly only

TABLE 5 SUSCEPTIBILITY OF RHESUS MONKEY KIDNEY CELLS TO POLIOVIRUS MEF-1 AT LOW MULTIPLICITIES OF INFECTION

Expt. no.

Number of PFU added to cells~

Time of adsorp- Virus % Number of tion to adcells exposed cells (min) s°rbed~

Multiplicity of infectionc (m)

Number of cells giving plaques

Fraction Calculaof cells ted multigiving plicity of plaques infectiond (P) (ml)

1

2 . 4 X 107 2.4 X 107

60 30

3.3 1.6

5.9 X 105 5.1 )< 105

1.27 0.73

4.9 )< 105 2.2 X 105

0.84 0.44

1.8 0.58

2

2.8 X 107 2.8 X 107

60 30

2.5 1.1

1.2 )< 106 1.2 X 106

0.57 0.26

3.2 X 105 2.9 )< 105

0.26 0.25

0.30 0.29

120 120 120 120 120 120

4.5 4.5 4.5 4.5 4.5 4.5

3.0 2.2 3.0 2.3 2.8 3.0

0.3 0.16 0.075 0.039 0.016 0.003

3.0 1.4 2.3 4.0 3.8 6.0

0.10 0.07 0.075 0.018 0.012 0.002

0.10 0.07 0.075 0.018 0.012 0.002

2.0 8.0 5.0 2.0 1.0 2.0

)< X )< X )< X

107 108 106 106 106 105

)< X )< X X X

108 106 106 106 106 106

)< X )< X )< X

105 105 105 104 104 103

Titer of virus suspension added was corrected for low plating efficiency as described in text. b Purified P3~-labeled poliovirus was used, and the percentage of ps~ adsorbed to cells was taken as an estimate of percentage of infectious virus adsorbed. Determined from ratio PFU virus adsorbed no. cells exposed to virus" d Calculated from the expression: P -- 1-e-m1 .

436

JOYCE TAYLOR AND A. F. GRAHAM

+1.0

.o K

such as a normal distribution; the resulting curve relating P and m would then be a composite of the curves m = 2, 5, 10, 15, and 20 of Fig. 5. The chance t h a t this resultant curve would be similar to P = 1 e - ~ is remote. I t is concluded, therefore, t h a t the cells of the m o n k e y kidney populations are uniformly susceptible to infection by a single particle of M E F - 1 virus.

S L O P E • 1.086

= 0.0 E O

/

O

0

-I.0

Correlation between Number of Cells Infected and Number of Cells Killed by Adsorption of Virus.

t9 O .J -2.0 -2.0

-I.0 LOS

I 0.0 m (observed

+1.0

multiplicity)

:FIG. 6. R e l a t i o n s h i p b e t w e e n log m ( m u l t i p l i c i t y of i n f e c t i o n d e t e r m i n e d e x p e r i m e n t a l l y ) a n d log m~ ( m u l t i p l i c i t y of i n f e c t i o n d e t e r m i n e d f r o m expression P = 1 -- e -ml (see T a b l e 5).

at the highest multiplicity; all other factors but the virus dilution were k e p t constant. The results are plotted in Fig. 5 where they are seen to fall reasonably close to the theoretical curve P = 1 -- e - % To test the goodness of fit of the data, a value of m = ml was calculated for each value of P in Table 5 by assuming the relation of P = 1 - e - " " . I f only a fraction, ], of the P F U adsorbed were effective in creating an infeeted cell, P = 1 - e -I~, where m is the observed multiplicity and m~ = ].m. Therefore, a plot of log m~ against log m should give a straight line with a slope of 1 and an intercept of log f. The straight line shown in Fig. 6 was fitted to the data, with such coordinates, using the method of least squares, and the slope of this line did not differ signifieantly from unity b y the t test. As would be expected the value of f was close to unity. The slope of unity indicates a reasonable fit to the distribution P = 1 - e -m shown in Fig. 5 and not to the other curves, and we can eliminate the possibilities of cell heterogeneity discussed under headings (2) and (3) of this section. I t is possible to envisage a situation rather more complex t h a n t h a t discussed under heading (2), in which the n u m b e r of adsorbed particles required to initiate infection of the cell follows another distribution,

The particles of a virus population m a y differ among themselves in their rates of adsorption to susceptible cells. However, as has been mentioned earlier in this paper, the correspondence in rates of adsorption of p3e and infectivity for labeled M E F - 1 virus indicates t h a t the m a j o r fraction of labeled particles is relatively uniform with respect to adsorption rate. On the reasonable p r e m ise t h a t our purified P3e-labeled virus is similar in chemical composition to t h a t of Sehwerdt and Schaffer (1955), most of the particles visible in the electron microscope should contain P~% Since purified poliovirus contains about 30 % R N A (Sehwerdt and Sehaffer, 1955), the great m a j o r i t y of the particles must contain R N A phosphorus, should become labeled during its intracellular formation, and, therefore, should adsorb to the cells. Whether all the particles adsorb could be determined directly with purified virus containing a specific protein label; our experiments on this aspect are so far inconclusive largely because of difficulty in introducing enough C 14 or S 35 specifically into virus protein. Nevertheless, it is fair to assmne t h a t m a n y more particles adsorb to a m o n k e y kidney cell monolayer t h a n give rise to plaques. This a r g u m e n t raised the possibility t h a t there m a y be an excess of cell-killing particles over infectious particles. I f such were found to be the case, evidence for a heterogeneous virus population would be provided. The reduction in cloning efficiency of a cell population was t a k e n as a measure of the n u m b e r of cells killed. Monolayers of m o n k e y kidney cells were exposed to a suspension of purified M E F - 1 virus for 60 rain-

PLAQUE ASSAY FOR POLIOVIRUS utes at 37 °. The lnonolayers were washed free of unadsorbed virus with PBS, suspended, counted, and plated (a) on fresh monolayers to determine the number of infected cells, (b) to determine the cloning efficiency of the population as described under Materials and Methods. The results are shown in Table 6. I t is clear from these results t h a t there is a close correspondence between the rates at which cells are killed and infected by M E F 1 virus. Similar results have been obtained with the N D V - H e L a system (Marcus and Puck, 1958). Apparently, any noninfectious particles that adsorb either do not have the ability, or the opportunity, to kill the cells. I t might be mentioned t h a t the observations presented here and in the previous section eliminate a possibility, suggested by Schwerdt and Fogh (1957), that the routine scoring of plaque assay plates after 4 days might give a low estimate of titer because of the very late appearance of additional plaques. If this were the case in our assay procedure, we should have expected a large deviation from the simple Poisson expression relating multiplicity of infection and formation of infectious centers, and might have expected a lack of correlation between numbers of infected and killed cells. DISCUSSION

The work described in this paper has permitted an inquiry into the problem of the ratio of physical to infectious particles in poliovirus. In the knowledge t h a t each cell of the monkey kidney monolayers is susceptible to infection by a single virus particle, there are two possible explanations to account for a 30 to 1 ratio of physical to infectious particles in an M E F - 1 virus suspension. Either there are t h i r t y times as m a n y adsorption sites as entry sites per cell, or there are t h i r t y particles incapable of infecting for every infectious one; the two possibilities are not mutually exclusive. The data have not permitted these two resulting hypotheses to be resolved. The terms " e n t r y " and "adsorption" sites, which have been used in this paper in their broadest sense without implying any mechanism, have recently been given some practical expression by Darnell and Sawyer

437 TABLE 6

CORRELATION BETWEEN NUMBER OF CELLS INFECTED AND NUMBER OF CELLS KILLED IN CULTURES INFECTED WITH POLIOVIRUS M E F - l N u m b e r of Time. °f % Cells % Expt. cells exnosed ausor • . % Cells Cells , P.u o n giving no. .oI virus , a killedb into virus (min) clones fected 3.08 X 106 3.6 X 106 2.4 )< 106

0 60 60

25 21 18

-16 28

-6 25

3.8 X 106 2.6 X 106 2.7 )< 106

0 60 60

16 10 12

-39 23

-29 22

a C l o n i n g efficiency as d e s c r i b e d in M e t h o d s . b Determined from the ratio c l o n i n g efficiency u n i n f e c t e d cells c l o n i n g efficiency i n f e c t e d cells X 100. c l o n i n g efficiency u n i n f e c t e d cells

(1959, 1960). These workers have shown that clonally selected populations of H e L a cells m a y v a r y markedly in their capacity to be infected with poliovirus, whereas RNA extracted from the virus will infect the clones with equal efficiency. An adsorption site is therefore an entry site if it permits effective release of viral R N A into the virusinfected cell. In view of this demonstration that the proportion of entry to adsorption sites can vary from one clone to another, it is at least theoretically possible that those particles of a poliovirus population that have been considered noninfectious may, in fact, be capable of infection under the proper circumstances. As yet, the possibility of heterogeneity in the virus population cannot be eliminated and this aspect of the problem is under present investigation. ACKNOWLEDGEMENTS T h e a u t h o r s are grateful to Mr. L. Pinterie for the electron microscope estimates, to Mr. O. H u t z i n g e r for technical assistance, to Dr. L. Siminovitch for m u c h helpful discussion, and to Mr. W. Reid for advice a b o u t statistical m e t h o d s . REFERENCES BACIIRACI¢, H. L., CALLIS, J. J., HESS, W. R., and PATTY, R. E. (1957). A p l a q u e assay for f o o t - a n d m o u t h disease virus a n d kinetics of virus r e p r o duction. Virology 4, 224-236.

438

JOYCE TAYLOR AND A. F. GRAHAM

DARb!ELL, J. E., JR., and SAWYER, T. K. (1959). Variations in plaque-forming ability among parental and clonal strains of HeLa cells. Virol~ ogy 8, 223-229. DARNELL,J. E., JR., and SAWYER,T. K. (1960). The basis for variation in susceptibility to poliovirus in HeLa cells. Virology l l t 665-675. DULBECCO, R., and VOGT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exptl. Med. 99, 167-182. I)UNi~EBACKE, W. ~I., and I~EAUME, M. B. (1958). Correlation of the yield of poliovirus with the size of isolated tissue cultured cells. Virology 6, 8-13. GRAI-I.~,M, A. F. (1959). Physiological conditions for studies of viral biosynthesis in mammalian cells. Bacteriol. Revs. 23~ 224-231. MARCUS, P. I., and PUCK, T. T. (1958). Host-cell interaction of animal viruses. I. Titration of cell-killing by viruses. Virology 6, 405-423. PARKER, R. C., CASTOR, L. N., and McCuLLOCH, E. A. (1957). Altered cell strains in continuous culture, a general survey. In "Cellular Biology. Nucleic Acids and Viruses," pp. 305-313, New York Academy of Sciences, New York. PUCK, T. T., MARCVS, P. I., and CIECIURA, S. J. (1956). Clonal growth of mammalian cells in

vitro. Growth characteristics of colonies from single HeLa cells with and without a "feeder" layer. J. Exptl. Med. 103, 273-284. REID, D. B. W., and MAcLEoD, D. R. E. (1954). The relation between dose and mortality for Salmonella dublin. J. Hyg. 52, 18-23. SC~rWERDT, C. E., and SCnAFFER, F. L. (1955). Some physical and chemical properties of purified poliomyelitis virus preparations. Ann. N. Y. Acad. Sci. 61, 740-750. SC~WERDT, C. E., and FOG~, J. (1957). The ratio of physical particles per infectious unit observed for poliomyelitis viruses. Virology 4, 41-52.

TAKEMORI, •., NOlVLURA,S., NAKANO,M., MORIOKA, Y., HENMI, M., and KITAOKA,M. (1958). Mutation of polioviruses to resistance to neutralizing substances in bovine sera. Virology 5, 30-55.

TAYLOR, J., and GRAHAM,A. F. (1958). Purification of poliovirus labeled with radiophosphorus. Virology 6~ 488-498.

TAYLOR,J., and GRAHAM,A. F. (1959). Studies on P3-~-labeled poliovirus. Trans. N. 17. Acad. Sci. [2] 21, 242-248. YOUNGNER, J. S. (1956). Virus adsorption and plaque formation in monolayer cultures of trypsin-dispersed monkey kidney cells. J. Immunol. 76, 288-292.