VIROLOGY 9, 546-563 (1959)

Host-Cell Interaction of Animal Viruses

II. Cell-Killing Particle Enumeration: Survival Curves

at Low Multiplicities” 2* 3

-4rcepted September 1, 1959

Single-cell survival curves were obtained for two different strains of New- castle disease virus (NDV) interacting with two mutant strains of 53 HeLn cells.

53-9 HeLa cells when treated with increasing concentrations of two different NDV strains, exhibit a one-particle-to-kill curve until approximately 95 y0 of the cells have been killed. Further increase in virus multiplicity results in more cell survivors than theoretically expected. This deviation from theo- retical behavior at moderately high multiplicities can be decreased by addi- tion of the virus in several small increments. In this way, the number of cell survivors of infection with a multiplicity of 9 NDV part,icles can be decreased by 25.fold.

A mutant subclone of HeLa 53-9 was isolat,ed which displays more uniform susceptibility to the lethal action of iYDV than the parental strain. The mu- tant, designated S3-9(IV), exhibits a curve which quantitatively follows one- hit kinetics down to a survival of less than one-tenth of 1 %.

Penetration of NDV is essentially complete in less than 5 minut,es at 3i” as measured by production of a lethal virus-HeLa cell complex, refractory to the viricidal action of the complete growth medium here employed.

Modified cell-killing procedures permit virus penetration at physiological temperatures and circumvent the cell-virus-cell agglutination reactions ob- served at these temperatures.

The Dulbecco plaque technique was modified in order to score the maximum number of KDV plaque-forming particles. NDV-Beaudette plaque tit,ers so obtained were tn-ice the usual value and showed excellent agreement with cell-killing-particle numbers as assayed on HeLa cells. The Beaudette and Vaccine strains of SDV display high cell-killing titers for S3 cells, 2 X lOs/cm” and 7 X 109/cm3, respectively, despite the fact that the plaque-forming t,itel

1 This research was supported by a grant from The National Foundat,ion. 2 Contribution No. 88 from the Department of Biophysics, Florence R. Sabin

Laboratories, Universit,y of Colorado Medical Center, Denver. 3 Aspects of some of these experiments have been reported elsewhere (Xlarcus,

1959a,b).

546

HOST-CELL INTERACTION OF rlNIMAL VIRUSES. II 547

on chick embryo cells of the former is 2 X 109/cm3, whereas that of the latter may be less than 5 X 104/cm3. Other assagable properties of NDV stock preparations are compared.

INTRODUCTION

Use of cell plating techniques for quantitative measurement of the lethal action of viruses on the reproductive capacity of mammalian cells has been described in earlier publications (Marcus, 1957, 1959a; Marcus and Puck, 1958a,b). The killing of HeLa S3 cells by the Beaudette strain of Newcastle disease virus (NDV-B) was shown to follow a one-particle- to-kill law for virus multiplicities up to 3 or 4. Thereafter, however, cell survival in excess of the one-hit relationship was observed.

This paper describes (1) a modified plating procedure which scores the maximum number of NDV plaque-forming particles, (2) procedural modifications which permit virus penetration at 37”, but obviate cell- virus-cell agglutination reactions, (3) low multiplicity survival curves of two mutant cell lines of HeLa S3, one of which exhibits a linear response to NDV killing over a wider range of multiplicities, (4) some factors affecting expression of cell killing, and (5) cell-killing-particle assays of two strains of NDV.

MATERIALS AXD METHODS

Cell Strains and Assay

Both cell mutants were subclones of HeLa S3. The first, S3-9, is indistinguishable from S3 in its reaction with NDV, but has more com- pact colonial morphology. The second, S3-9(IV), was derived from S3-9 through two successive clonal isolations and has a diffuse colonial morphology like the original S3 (Puck et al., 1956). Lag period, genera- tion time, plat,ing efficiency, and, to a first approximation, nutritional requirements of both mutants are the same as those of HeLa S3 (Puck et al., 1956; Fisher et al., 1959). X-Ray and ultraviolet irradiation survival tests, as well as chromosomal delineation studies are in progress.

Standard procedures were used for maintenance of cell stocks and single-cell plating (Puck et al., 1956; Marcus et al., 1956). A new modifi- cation of the t,rypsinization technique was employed (Puck et al., 1958).

Virus Strains

The Vaccine (Blacksburg) strain of Newcastle disease virus (NDV-V) was kindly supplied by Dr. F. B. Bang (Liu and Bang, 1953; Bang and Warwick, 1957). The Beaudette strain (NDV-B) and the standard

548 MARCUS

procedures of virus stock preparation were used as previously described (iLiarcus and Puck, 1958b). Approximately lo3 egg-infectious or plaque- forming particles of the Vaccine and Beaudette strains, respectively, were used to inoculate each lo- or 11-day-old egg. Both strains of virus grow equally well in the allantoic sac’ of eggs (Liu and Bang, 1953).

Virus Assays

Plaque-forming particles (PFP). Assays were performed by the follow- ing modification of Dulbecco’s plaque technique (Dulbecco, 1952; Dulbecco and Vogt, 1954), which maximizes the number of plaque- forming particles by (1) extending the attachment period of iYDV on chick embryo cell monolayers, (2) replacement of the phosphate-buffered saline (PBS) with Attachment Solution, and (3) use of a lowered tem- perature of ntt,achment to delay new virus release:

a. Chick embryo cell suspensions were prepared as described by Dulbecco and Vogt (1954). Monolayers from first- or second-passage cells were grown on loo-mm pressed-type petri dishes in Nutrient Solu- tion (93 %) (Marcus et al., 1956) and calf serum (7 %) wit,h penicillin (200 U,/cm3) and streptomycin (200 pg/cm”), at 37” in an atmosphere of 5 % CO, in humidified air. All operabions subsequent to preparation of the cell monolayers, and preceding final incubation for plaques, were performed at 20-22” with solutions previously brought. to t,his ambientj temperature.

b. Cell layers mere washed two times with s-cm3 amounts of Attach- ment Solution (Marcus and Puck, 1958b), a solution which eliminates the delet,erious effect of PBS on the integrity of the cell sheet, and thus on plaque development.

c. Virus was diluted in Attachment Solution and layered over t)he washed monolayers as 0.5-ml volumes by pipetting 0.1 ml of an appro- priat’e dilution directly into 0.4 ml of previously added Attachment Solution concentrated in one spot by tilting the plate.

d. Cell layers mere exposed to virus for as long as 340 minutes. I’ro- longed attachment periods were accompanied by periodic tilting of t,he plates, every 30 minutes, to distribute the virus evenly and prevent drying out of high spots in the cell sheet.

e. At the end of the at’tachment period a 1. % agar overlay, containing Nutrient Solution, calf serum, and antibiotics in concentrations as described above, was added to the monolayers. (Chick embryo extract in the overlay was discontinued when cytotoxic effects were observed on several occasions.)

HOST-CELL INTERACTION OF ANIMAL VIRUSES. II 549

NDV-6 Cl: Chick Embryo Cells

I 0 50 100 150 200 250 300 350

ATTACHMENT TIME (MINUTES)

FIG. 1. The time course of attachment of NDV-Beaudette (Cl) to chick embryo cell monolayers. The circles and dots represent experiments separated in time by five months. Each point represents the mean plaque count of triplicate platings on loo-mm dishes. Agreement between replicate counts was within sampling error, f20 %.

f. After 3 days’ incubation, plates were stained with 1: 10,000 neutral red in Attachment Solution for 1 hour to permit better visualization of the plaque areas. Maximum contrast between cell sheet and plaque area was achieved by an additional overnight incubation of the stained cell layer after removal of excess staining solution. Plaque areas continue to increase in size, at a rate governed by the agar concentration in the overlay. No significant increase in plaque count results from still further incubation, although plaques at the periphery of the plate become easier to score after one more day.

Representative results from this procedure are illustrated in Fig. 1. The maximum yield attained is more than double that obtained with the usual procedure. The absence of new virus production at 22” reported for the NDV-HeLa cell system (Tyrrell, 1955) makes possible this prolongation of attachment time.

Egg-infectious particles (EIP). Egg ID,,, titers were calculated by the method of Reed and Muench (1938) using 6 eggs per &j8 log dilution, and were converted to infectious particles by the Poisson distribution formula whereby 1 EIDSO = 0.693 infective particle. Infected eggs were

550 MARCUS

determined by demonstrating the presence of virus hemagglutinin at lo+, 10p3, or 1O-4 dilutions of allantoic fluid.

Hemagglutinating particles (HAP). Titration was by means of t’he pattern technique (Salk, 1944) using two-fold serial dilutions of virus in phosphate-buffered saline at, room temperature. Pooled chicken red blood rells were present in each tube in a final concentration of 7.2 X IO6 cells per cubic centimeter. Under these conditions a positive pattern in t,he last tube reflects the presence of 3.4 X lo5 hemagglutinating particles, according to the assay procedure of Levine et al. (1953).

Cell-killing particles (CKP). Single-cell survival experiments were performed as reported earlier (Marcus and Puck, 1958b), except that virus at’tacahment to cells was carried out. at O-2”. After 30 minut,es the temperature was raised by dilut,ing t,he virus-cell complexes with at least 100 volumes of Attachment Solution at 37”. The complexes were t,hen held at, 37” before plating into Complete Growth Medium for colony development. Penetrat,ion studies with the Beaudette and Vawine strains reveal that less than 10 minut’es at 37” suffice to form lethal virus- cell complexes completely stable to single cell platings in the viricidal Complete Growth Medium4 (see Experimental Results).

Determination of the absolute cell-killing particle multiplicity (mck,,) is calculated from the fract,ion of survivors (C,‘C,) and the Poisson dis- tribution, where

e-mckp = (‘/Co (1)

The cell-killing particle (CKP) t,iter of the original suspension is then enumerated as follows :

CKI’ _ mchh ’ a (2)

where CKP = concentration (particles/cm3) of virus capable of killing cells; h = host cell concentration (cells/cm3) during virus att,achment; n = reciprocal of the virus dilution; a = fraction of total virus attached. This relationship is valid only for the exponential portion of the cell survival curve (1); t,herefore, several dilutions of a virus suspension are t’est,ed in such an assay, and the CKP concent’rat,ion determined from the values which fall on the linear part of the curve.

Determination of a, t’he per cent, attachment’ to cells, was measured

4 The half-life of NDV-B plaque-forming particles in Complete Growth Medium at. 37” is about 5 minutes (Cieciura, 1958) or less (unpublished observations).

HOST-CELL INTERACTIOX OF ANIMAL VIRUSES. II 551

for the Beaudette strain by plaque formation; but for the Vaccine strain which displays poor plaque-forming efficiency (see below), it was neces- sary t,o use the less precise though more rapid hemagglutination test. The uptake of hemagglutinating particles of the Vaccine strain is idenbi- cal to that of the Beaudette strain under the same conditions, i.e., 75 % of the hemagglutinating particles are adsorbed by a suspension of 1.0 X lo7 cells per milliliter during a 30-minute attachment time at O-2”.

Since parallel measurements with the Beaudette virus strain always yielded agreement between virus uptake as measured by hemagglutina- tion and plaque titration under these standard conditions, t’he value of 80 % attachment for the 2-37” sequence, as determined with the more accurate plaque technique, was also used in the calculations of a for the Vaccine strain.

EXPERIMEETAL RESULTS

Cell-killing Kinetics at Low Multiplicities

The Vaccine strain. Results from typical single-cell survivor experi- ments are presented in the protocol of Table 1. Comparison of the effects of the same lot of virus, on the two cell mutants S3-9 and S3-9(IV), is shown by the survival curves in Fig. 2. The cell-killing particle multi-

TABLE 1

RESULTS FROM A TYPICAL SINGLE-CELL SURVIVAL EXPERIMENT: HELA S3-9(IV)-NDV(VAcCINE) INTERACTION

RWC- tion tube

A B C D E

‘irus dilution froy$ck

-

\

-

-

1:700 1:350 1:192 1:96

No virus

Survivors: individual colony counts (C) and mean

200 64, 90, 73, 83 = 77.5 0.388 0.95 1000 226, 228 = 227 0.227 1.50 1000 32, 47, 39, 28 = 36.5 0.0365 3.35 5000’ 9, 12,5,6, 13, 11, 10,9 = 9.3 0.0018 6.30 100 120, 111, 106 = 112 1.12 - - -

-

Stock titer (CKP/nW

8.3 X lo9 6.6 x 109 8.0 X lo9 7.0 x 109

- a CKP = [(mck,,)(h) (d)/a]; h = 1.0 x lO’/ml; d = reciprocal of virus dilution;

a = 0.80 (see Materials and Methods). * Inocula of 5000 cells are plated into lO@mm diameter petri dishes with 20

ml of Complete Growth Medium to obviate cross-infection (Marcus and Puck, 1958b).

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CELL-KILLING PARTICLE MULTlPLlClTY (M,,,)

1

1

3 0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5

VIRUS CONCENTRATION i lo3 (V)

FIG. 2. The survival of HeLa S3-9 and S3-9(IV) as a function of the concentra- tion of Newcastle disease virus-strain Vaccine. Virus concentrations (V) on the lower abscissa have been converted t,o cell-killing-particle multiplicities (mckp) on the upper abscissa, where V necessary to reduce the initial population to e-1 (37 ?JO survivors) results in an attached n8ekp = 1.0.

plicity (mckp) is 1.0 when the virus concentration (V) is 0.001625, i.e.,

a 1:615 final dilution of t’his particular virus stock. It is important to note in Fig. 2 that this same value is obtained for the virus cell-killing titer when either S3-9 or the S3-9(IV) cell mutants are employed, since the survival curves for both cells are superposable for low virus multi- plicities. However, the curve for the ST9 cell begins to flatten at a virus

HOST-CELL INTERACTION OF ANIMAL VIRUSES. II xi3

multiplicity greater than 3, and that for S3-9(IV) obeys the one-hit relationship until a multiplicity of 6 has been reached.

Representative experiments illustrating the reproducibility of cell survival curve determinations are shown in Fig. 3, where three independ- ent runs on one lot of NDV-V are plotted.

l’he Beaudette Strain. Survival curves for both cell mutants exposed to NDV-Beaudette are compared in Fig. 4 where it is clear that exactly the same relationships obtain for the two cell mutants, as was the case

0.002 -

0.001 I I I 0 2.5 5.0 7.5 lo.0 12.5 15.0

VIRUS CONCENTRATION X lo3 (‘4)

FIG. 3. Demonstration of the reproducibility of cell survival curve determina- tions: The survival of HeLa S3-9(IV) as a function of low concentrations of NDV- Vaccine. The different symbols represent three independent runs on one lot of virus carried out over a period of seven months. Each point on the curve repre- sents the mean fraction of survivors determined from cell platings done in tripli- cate, and with dilutions chosen to produce about 100 colonies per plate.

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CELL KILLING PARTICLE MULTIPLICITY IM,,,)

2 0005 + ‘0,

Y

6

\

0 001 0

Heto S3-9(IV)

0 0005

* I I I

oooos J 0 0 01 0 02 0.03 004 0.05 0.06 0 07

VIRUS CONCENTRATION (V)

FIG. 4. The survival of HeLa 53-9 and 2%9(N) as a function of the concentra- tion of Newcastle disease virus-strain Beaudette. The concentrations of virus (I-), plotted on the lower abscissa have been converted to cell-killing-particle multiplicities (w&k,,), where an average of &kp = 1 is obtained when V reduces t,he original population to e-l, or 3i $X0 survivors. Cell-killing-particle mnltiplici- ties are plotted on the upper abscissa.

n-ith the Vaccine strain. Also, the same degree of reproducihilit,g of survival curves with the Beaudette strain is obt’ained as wit.h the Vaccine strain of virus.

Factors Aflecting Expression of Cell Killing

Experiments demonstrate that the rnt,e of virus attachment t,o both cell mutants is the same. In a representative experiment,, when the Beaudette strain of KDV was added to 1.0 X lo7 cells per cubic centi- meter at an input of about 6 PIT under the conditions of the dual temperature-dilut’ion sequence, i.e., at)tnchment at O-2” with the result-

HOST-CELL INTERACTIOK OF ANIMAL VIRUSES. II 555

ing virus-cell complexes diluted and held at, 37” for lo-15 millutes, the fractions of plaque-forming particles attached to HeLa B-9 and SS-9(W) cells were 79.2 and 82.5%, respect’ively. Thus, some reaction(s) subse-

quent’ to virus att’achment, and related t)o the binding of a relat’ively large iimllher of virus particles, appears to be limiting t,he killing of M-9 cells at this multiplicity (cf. Fig. 3).

Experiments were carried out to determine whet,her t,he surviving fraction of cells at mLli,, = 6 was influenced by the length of time spent by the virus-cell complexes in the nonviricidal Mtachment Solution (at 37”) prior t,o plat)ing in the viricidal growvth medium. In a typical experiment, (SDV-R)-cell complexes were formed separately at O-2” for each cell strain, rapidly diluted by adding Att,achment Solution at 37”, and at, KlkJUS times aliquotjs of this cell suspension were removed and

quicskly placed into growth medium and plated for survivors. It was cstnblishrd that 5 minutes in Atjtachment Solution at, 37” is sufficient to obtain the minimum possible surviving fraction for either cell mutant. Thus, if the period in Attachment Solut,ion at 37” was iwreased to 1 hour, no further decrease in colony formation occurred. Since all cells which rcwiw I to 3 let,hal particles are killed, it becomes important to det,ermine whether the abnormally high cell survival whicbh occurs at high virus multiplicities is dependent on the degree of simultaneity of attachment by multiple virus part,icles. Experiment,s were performed in which a series of virus additions separat,ed by definite time intervals was made to a suspension of host cells. h t)otal mClip = 9 was added to

TABLE 2 Conwamsox OF THE OBSERVEI) ASD THEORETICAL FRACTIOS OF HlsL.4 93-9 CELL

SI-R~IX~~R~ AFTER EXP~SURI~ ~0 SIMI~LTANEOUS ANI) Pruim ~~I)ITIOKS OF SEWCASTLE DISEASE VIRUS (VAUXNE)

Conditions of CKP addition

Total cell-killing-particle multiplicity (ra,,k,,)

3.0 6.0 9.0

Simultaneous Pulsed=

0.062 0.023 0.010 0.058 0.002i 0.00037

Theoretical survivalh 0.050 0.0025 0.00012

n First attachment multiplicity, nl,kp = 3; second, rnckp = 3 + 3 = 6; and third, VLcky, = 3 + 3 + 3 = 9.

* From C/CO = e-cb, where C/CO is the fraction of cell survivors [cf. expression (111.

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TABLE 3

THE CELL-KILLING PARTICLE ASSAY OF NDV-VACCINE AND BEaUDETTE ON HELA CLONES s3-9 AND s3-9(IV)

Virus stock Host cell Virus strain (date of

preparation) s3-9 83.B(IV)

Vaccine 3/58 7.4 f 0.5 x loga 8.0 Ik 0,s x 109 10/58 7.1 f 0.9 x 109 7.2 h 1.5 X lo9

Beaudette d/54 2.2 f 0.1 x log 1.9 f 0.3 x 109 o/57 2.5 f 0.6 X lo9 l/58 2.1 f 0.4 x 109 2.3 f 0.4 X lo9

n CKP titer per millilit,er: mean and standard deviation of three independent, assays.

SS-9 cells in three equal virus additions. Between each addition of virus at O-2” the virus-cell complexes were diluted in Attachment Solution at 37” and held for 10 minutes. The complexes were then reconcentrated by centrifugation for another input of virus at t,he lower temperature, and the cycle repeated until a total of 9 cell-killing particles had been attached. Cell survivors were determined after each pulse of virus attach- ment and penetration. The results from a representative experiment,, as tabulated in Table 2, demonst’rate that the excessively high fraction of s?,-g cell survivors at m&B = 3-9 (cf. Fig. 2) is a direct’ consequence of the simultaneous attachment of virus and that if particles are pulse- attached in increments of 3, then the theoret,ically expected degree of cell killing is essentially realized. Further analyses of T\‘DV penetration kinetics are in progress.

--Issay of Diferent Properties of Particles in NDV Stocks

Table 3 presents the number of cell-killing particles in Beaudette and Vaccine strain stock preparations as determined with both S3-9 and S3-9(IV) cell mutants. Although both cell types yield the same result obviously the S3-9(IV) strain is superior for such titrat,ions because of t,he greater range over which its response is linear.

In Table 4, CKP assays are compared with t,he tit,ration values for hemagglutinating, egg-infectious, and plaque-forming particles per- formed simultaneously on NDV suspensions. The data of Table 4 demon- strate the essential equivalence of Beaudette strain HAP, CKP, and

HOST-CELL INTERACTION OF AUMAL VIRUSES. II 557

PIP, particularly when t’he last-named particles are measured by the modified attachment method described above. The slightly low egg- infectious particle titer is thought to reflect unfavorable attachment conditions in ouo.

In marked contrast to t’he Beaudette strain assays, the data in Table 4 show that plaque formation for a freshly harvested stock of XDV- Vaccine detects about lo5 times fewer particles than the number of CKP. Plaque formation appears to be very labile in this virus and virt,ually disappears even when the virus is stored at temperatures of -50” for a few months. It is not uncommon to obtain plaque-forming particle t,iters of less t’han 103/cm3 from freshly harvested EDV-V stocks. This lability may reflect a special molecular requirement for preservation of plaque-forming ability by this strain, since several other st,rains of XDV regularly maintain a high yield of plaques under identical con- ditions of storage and plating. The egg infectivity titer of the Vaccine strain is about tenfold less than the cell-killing titer, a relationship which has been observed with two other independent assays of these two properties. It is noteworthy that t’he cell-killing particle titer is high and constant in both virus stocks employed (Table 3) and equals the titer of hemagglutinating particles (within the uncertainty of that measure- ment) despite the large differences in plaque-forming and egg-infectious titers of these suspensions.

It should be noted that relatively constant CKP titers of different lot,s of the Beaudette and Vaccine strains are obtained, and t,hat the Vaccine strain usually exhibits a higher value (Table 3). Preliminary results of CKP titers performed on a slowly eluting strain of l%DV also show essentially equivalent assays from lot to lot.

TABLE 4

COMPARISON OF TYPICAL CELL-KILLING (CKP), PLAQUE-FORMING (PFP). EGG-INFECTIOUS (EIP), AND HEMAGGLUTINATING (HAP) PARTICLE

TITERS OF BEAUDETTE AND VACCINE STRAINS OF NDV (PER MILLILITER)

NDV Strain and stock (date)

CKP PFP EIP HAP

Beaudette (l/58) 2.1 X 109 1.9 x 109 1.0 x 109 3 x 109 Vaccine (10/58) 7.1 X 109 5.1 X lo4 5.2 X lo8 7 x 109

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The quant,itative detect,ion of cell-killing particles introdwes an ad- ditional criterion by which virus activity may be assessed. The forego- ing experiments demonstrat,e that stock preparations of the Beaudette strain of NDV possess equivalent cell-killing, plaque-forming, and hemagglutinating activit,y, whereas successful egg infect’ivity apparently occurs with a probability of about one-half for these particles, under the conditions here employed.

Cell-killing part,icle assays with the Vawine strain reveal a striking lack of parallelism with plaque-forming particle assays. The following considerations would appear to account for these observations: In principle, plaque formation should proceed optimally under wnditjions of rapid host cell destruction aided by short latent periods, large yields of stable infectious particles, and rapid diffusion of virus. The work of

Bang and Warwick (1957) demonstrating the relatively slow destruction of tissue-cultured chick embryo fibroblasts by the “avirulent” Vaccine s;train compared to the rate of cytopathogenicity produced by a virulent strain of NDV suggests t,hat failure of one or more of the necessary condi- tions may contribute to the low yield of plaque formation from standard XDV-Vaccine preparat’ions. In wntrast, the scoring of a cell-killing particle requires only that, it kill its original single host cell, and thus, the need for subsequent production of new infectious virus to propagate successive cycles of cell destruction is obviated. Whether cell killing requires relatively less of a virus particle’s integrity than that required for formation of new infectious virus as is t,he case in some bacteriophage, or whether virus multiplication i!: a prerequisite to cell killing remains

t.o be determined. Vaccine strain virus may apparently lose 99.99 % of its plaque-forming abilit,y after three months’ storage at -50”. During these same t’est intervals, and for a further test period of more than :I year, killing-particle assays remained stable at 7 X log CKP per cubic: centimeter. The Beuudette st,rain exhibits stabilit)y of both plaque forrnn- tion and cell killing under such conditions. In cwntrast to the stability of NDV hemagglutinin, the cell-killing property, like that of plaque formation, is readily inactivated by heat or ultraviolet, radiation (Mawus and Puck, 1958b), although inactivation by t)he latter agent still leaves :I particle which can induce a refractory state in the HeLa cell to superin- f&ion by active virus (Marcus, 1959b).

It is of interest that the ‘Lavirulent” nature of the Vaccine strain as demonstrated so clearly in adult cahickens, chicks, and mass tissue cul-

HOST-CELL INTERACTION OF AXIMAL VIRUSES. II 559

tures of chick embryo fibroblaats by Bang and co-workers (Liu and Bang, 1953; Bang and Warwick, 1957) is not reflected in these studies of virus interaction with single HeLa cells since, as with it)s virulent counterpart-the Beaudette strain-the attachment of a single particle of NDV-Vaccine prevents even one cell division (Marcus and Puck, 1958b).

In assays of three different XDV-Vaccine stocks the egg infectivity titers were consistently less by a factor of about 10 than the cell-killing particle titer. If this difference is real, as reports from the literature indicate,5 then most of the members of XDV-V populations are probably unable to initiate a sustained infective process and can be detected only under conditions where an abortive infectious cycle after t,he initial virus-cell reaction can still be scored, e.g., as a lethal effect on t,he cell.

The number of cell-killing particles in stock preparations of the t,wo virus strains studied, and from preliminary results, also for a third strain, tend to be constant and characteristic for a given strain: for example, three different lots of the Beaudett,e strain prepared over a four-year period were found to contain essentially equal numbers of cell-killing particles (Table 3). These result’s most likely stem from the apparent stability of cell-killing-particle activity, and t,he standard growth conditions used to produce and harvest the virus.

The dual temperature-dilution sequence described here serves t’wo useful purposes: It permits formation of a low temperature virus-cell complex which when kept at O-2” remains stable for at least 4 hours, during which the complexes can be subjected t,o various experimental manipulations without any drop in the plating efficiency of cells or change in the fraction of survivors. It also, for reasons which are not, yet clear, circumvents the cell agglutination reactions which otherwise occur if virus attachment takes place entirely at 37”.

It should be possible to determine the attachment kinetics for any virus with cell-killing capacity even though it may not produce plaques, agglutinate red blood cells, or produce overt disease responses in animals.

The rate at which NDV becomes refractory to the serum-contained viricidal constituents of Complete Growth Medium has been demon- strated to be so rapid as to require less than 10 minutes at 37”. This is consistent with the findings of Baluda (1957), who reported that SDV

5 Egg infectiousjo titers as averaged from values in the literature for this strain vary between 108.5 and lo”.0 per cubic centimeter of allantoic fluid (Liu and Bang, 1953; BurnRtein and Bang, 1958).

560 MARCUS

infection leading to new virus production could not be blocked by the addition of specific antiserum after 7 minutes at most. These results contrast sharply with the relatively slow rate at which a serum-stable state is reached in t’he poliovirus-HeLa (Payne et al., 1958), or influenza virus-chick embryo cell (Burnet and Lind, 1957) systems, where a half- life of about 30 minutes is report’ed. Whether the very active virus en- zyme systems of NDV contribut,e to this rapid rate of penetration re- mains to be determined.

The present experiments also demonstrate that different mutant, mammalian host cells can arise which, although sensitive to killing by a single virus particle, display a markedly different response to virus action when challenged by a high virus multiplicity. The kinetics of SDV-B attachment to the two mutant strains of HeLa are experi- mentally identical. Abortive penetration induced by the simultaneous addition of virus at the higher multiplicities is presumably responsible for survival of the multiply infected cells in the one case (S3-9 as host), whereas completed penetration results in t,heir death in the other [S3- 9(IV) as host]. This type of response recalls t’he behavior of cell-associ- at’ed NDV during the latent period (Rubin et al., 1957), t’he nonrecover- able and apparently non-cell-killing NDV of Prince and Ginsberg (1957), t,he “lost” poliovirus of Payne et al. (1958), and the bound infectious virus (influenza) of Ackermann et al. (1955). Furt’her st’udies are re- quired to elucidate abort,ive virus-host cell interaction. These con- siderations make clear t,hat, in contrast to the simpler situation which usually obt)ains in bact,eriophage systems, the terms “sensitivity” or ‘+esistance” are inadequate to describe mammalian cell response to viruses. The property of an increased range of susceptibility possessed by HeLa S3-9(IV) appears genet’ically stable, since over sixt,y genera- tions of reproduction have occurred from the time of isolation and no alt,erat’ion in behavior has been observed.

The present demonstration that differences occur in the pattern of virus interaction with “sensit,ive” HeLa cell strains, recalls earlier work from this laboratory, in which it was shown that resistance to destruc- tion by NDV among “resistant” HeLa mutants also was not absolute (Cieciura et al., 1957; Cieciura, 1958; Puck and Cieciura, 1958), as it often is in bacterial virus systems. Ot,her workers, using these cell-plating techniques, have shown that c*omplexities similar to those described by US with NDV (Marcus, 1957, 1959a, b; Marcus and Puck, 1958a, b)

HOST-CELL INTERACTION OF AXMAL VIRUSES. II 561

also govern the interaction of S3 cells with poliovirus (Vogt and Dul- becco, 1958; Vogt, 1958; Darnell, 1959).

The present experiments appear to hold implications of importance for the problem of animal virus detection in clinical and experimental situations. Different mutant cell strains may function differently as indicator systems for virus detection. Attention should be directed toward selection of cell mutants displaying a linear response over a maximum multiplicity range to virus killing. Finally, the use of clonal stocks like S3-9(W) with a larger range of one-hit response to virus in- activation, and the principle of pulsed-virus attachment, makes possible more efficient search for true genetically resistant cell mutants (cf. Puck, 1957) since surviving colonies of virus action will be less con- taminated with those of cells whose resistance is only physiological, and hence temporary (Marcus, 195913). These studies are continuing.

ACKNOWLEDGMENTS

The author wishes to express appreciation to Professor Theodore T. Puck fol helpful suggestions and discussions, and to Mr. George Barela for his competent preparation of plates of chick embryo cell monolayers.

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