Homologous interference by ultraviolet-inactivated Newcastle disease virus

VIROLOGY

4, 72-96 (1957)

Homologous

interference Newcastle

by Ultraviolet-Inactivated Disease Virus’s 2

M~HCEL Kerckof

Laboratories

of Biology,

A. BALXJDA~

California California

;1ccepted

April

Institute

of Technology,

Pasadena,

25, 1957

Within 6 minutes after the adsorption of one ultraviolet-inactivated particle of Newcastle disease virus to a lung cell, the latter becomes unable to support the production of progeny virus by homologous active virus adsorbing later. There occurs an exclusion which operates on an all-or-none basis. The rapidity with which the interfering reaction is induced depends upon the number of inactive particles that have been adsorbed. However, in a fraction of the cells interference can be overcome; these cells can become yielders of new virus with a small probability per superinfecting active particle. Exposure of the interfered cells to specific anti-Newcastle disease virus (NDV) serum eliminates exclusion; interference becomes insusceptible to the antibody in 50% of the cells 30 minutes after the attachment of the ultraviolet-inactivated (UVI) particles. The resistance to infection disappears 26 to 60 hours after exclusion has been induced; the cells then behave like normal cells. The type of exclusion studied here, brings about the destruction of the superinfecting virus. A cell where interference has been removed after superinfection with active virus must be infected a second time in order to yield progeny virus. The results suggest that this type of interference occurs at the cell surface. The quantitat,ive aspects of this phenomenon are discussed. INTRODUCTIOS Very little quantitative information on interference is available from experiments performed with animal viruses. It has been suggested by Fazekas de St,. Groth and Edney (1952) that 1 particle of influenza virus 1 Submitted to the California Institute of Technology in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 2 Aided by a grant from the American Cancer Society. 3 Present address: Department of Microbiology, Research Institute, City of Hope Medical Center, Duarte, California. 72

INTERFERENCE

WITH

NEWCASTLE

DISEASE

VIRUS

73

heated at 56” for 60 minutes may be suthcient to inhibit the production of new virus by superinfecting, active, heterologous influenza virus which are adsorbed later. However, one disadvantage for a more detailed quantitative analysis of the interference phenomenon is that influenza virus heated at 56” interferes slowly and incompletely (Henle, 1950; Fazekas de St. Groth et al., 1952; Horsfall, 1954). Ultravioletinactivated influenza virus appears, on the other hand, to exclude very rapidly both homologous and heterologous active virus in all t>he cells to which the virus has adsorbed (Ziegler et al., 1944; Henle and Henle, 1944). It has even been reported by Henle et al. (1947) that ultraviolet-inactivated (UVI) influenza virus added after the active homologous virus could suppress the multiplication of the active particles. An experiment carried out by H. Rubin in this laboratory first suggested that inactive Newcastle disease virus (NDV) interfered with the multiplication of active NDV in cultures of lung epithelium. This led to the present detailed investigation of the phenomenon of interference using UVI homologous virus as the interfering agent. In the present work NDV and chicken embryonic lung cells have been used as the virus-host system. The advantages of this system have already been pointed out by Franklin et al. (1956). MATERIALS

AND

METHODS

The following abbreviations are used: HA = hemagglutinating unit; LA-YE = Earle’s saline to which have been added 0.5% lactalbumin hydrolyzate and 0.1% yeast extract; NDV = Newcastle disease virus; PBS = phosphate buffered saline; PFU = plaque-forming unit; RBC = red blood cell; RDE = receptor-destroying enzyme; UVI = ultraviolet inactivated. The methods for preparing media, making monolayer cultures, and assaying the virus and the infected cells were the same as those described by Franklin et al. (1956). Virus. Two strains of NDV were used. Some of the earlier work was carried out with the Beaudette strain (Bang, 1948). This strain was soon replaced by the strain L-Kan 1948 (Hanson et al., 1949) because of the latter’s greater heat stability and capacity to produce higher titers of new virus. Viral stocks. Stocks were prepared by inoculating into the allantoic cavity of lo- to 11-day-old chicken embryonated eggs 0.2 ml of a viral

suspension containing approximately 5 X 10” I’I’IJ. After --L8hours the eggs mere chilled and t,he allantoic fluids harvested. The infective fluids were centrifuged at low speed followed by a high speed run in a Spimo model I, ultracentrifuge at an average force of 45,000 g for 30 minutes. The pellet was resuspended in I’RS and after standing overnight at do, the viral suspension was again centrifuged at low speed. The final supernate was then used as the immediate source of virus and stored at 4”. E’resh stocks were prepared about every third week. The stock titers varied from 2 t,o 8 X 10y I’I’IJ wit,h a 1’FIJ:HA ratio of about 3 X IO”. Hemagglutination titration. HA titrations were carried out) by t’he Salk pattern technique (Salk, 1944) with some modifications designed t’o destroy HA inhibitors and viral enzymes suggest,ed by Granot? (1955). A volume of 0.1 ml of a virus suspension was diluted into 0.7 ml of PBS and 0.1 ml of 0.1 N KaIO, was added. This mixt’ure was adsorbed for 30 minut)es at 1°C and any excessperiodate n-as neutralized with 0.1 ml of 40 per cent of glucose. Serial two-fold dilutions of the mixture were made in PBS and t,o 0.5 ml of this suspensionwas added 0.5 ml of 0.25 per cent washed chicken erythrocytes in PBS. Patterns were read after :{ to -4 hours of standing at 4°C. The readings of +, f or - were read as in Salk’s met,hod; however, f inst,ead of + was taken as the end point. Where + proved to be the observed end point followed by -, rrt was taken as the geometric mean of t’he two values. C’1’1 z,irus. The virus was irradiated by exposing 2.0 or 2.5 ml of the viral suspensioil in an open GO-mmpetri dish to t,he UV radiation from a Westinghouse germicidal lamp with a window 10 camlong and 5 cm wide. The light intensity at the sample was of the order of 20 ergs/’ mnG/sec of which 80% was emitted at the wavelength of 2537 A. The suspensionswere shaken every 40 seconds to insure proper mixing. An average inactivating dose of 15 hit,s per virus particle was given through most of this work (1 hit, = 37 % survivors). i2ntiserum. Rabbits were inoculated over a period of 2 months with fourt’een alternating imravenous and intra,muscular injections of 0.5 ml of active viral stock. The serum was obtained by cardiac punct’ure 1 week aft,er the last, injection. Antibodies to normal chicken embryonic tissues were removed by incubating the serum with about lox suspended chicken embryonic cells/ml for 7 hours at 37”. The cells were then c*ent,rifuged and the serum kept froze11at - 18”. This serum had a /< value (Dulhecco et al. 1956) of 30. At a dilut,ion

INTERFERENCE

WITH

NEWCASTLE

DISEASE

VIRIJS

75

of 1: 100 in PBS, it reduced the infectivity of a given amount of NDV to 1% in 15 minutes at 37”. The serum had no cytotoxic effect, when placed undiluted on monolayers. General

Experimental

Procedures

All the interference experiments performed may be summarily broken down into two general types which will be called infection on the plate and infection in suspension. Iqfection on the plate. Olle ml of the UVI virus inoculum was deposited on t’he cell layer adhering to the glass surface of a culture plate. After a definite time of adsorption, the inoculum was removed, and then with warmed PBS, the cells were washed free of unadsorbed virus. The cells were then subjected to whatever treatment the experiment called for, i.e., treatment with serum, incubation, etc. The cells were subsequently superinfected with a second inoculum of active virus. Aft,er having removed the unadsorbed free virus by two additional washings, a volume of 5 ml of Versene was added to the cell layer where it was permitted to act for 5 minutes. The resulting cell suspension was then pipetted several times until a preparation consisting of single cells with no more than 10 % of doubles was obtained. The cells were again washed three times in PBS by centrifugation and resuspended in PBS lacking Ca++ and Mg++ t’o give a final concentration of approximately lo6 cells/ml. The total number of cells present was determined by direct count in a Keubauer counting chamber. The cells were serially diluted in PBS to find the concentration which gave about 100 infective centers per plate when assayed by the plaque technique. The tube containing the undiluted cell suspension was centrifuged once more to sediment all cells and the supernatant fluid was in turn assayed to determine the amount of residual free virus. The difference in plaque counts between these two samplesgave the number of cells that yielded new virus; this fraction of cells will lat,Lr on be referred to as yielders. All plaque assays were carried out in duplicate. Infection in suspension. In this case a suspensionof uninfected single cells was first obtained with Versene. The cells were then centrifuged and resuspended for a given time in 1 ml of UVI virus. During that time the cells were shaken frequently to prevent clumping. Following adsorption of the virus the cells were washed by centrifugation in PBS.

76

BALUDA

Superinfection with active virus was performed in a similar manner. From then on, the steps were identical with those carried out in the experiment with infection on the plate. One-step growth curves were obtained by diluting the yielders into LA-YE or into LA-YE plus 10% ox serum and 5 % embryo extract in earlier experiments. The maximum yield of new virus per yielder was obtained when the infected cells were diluted in LA-YE medium to a final concentration of about lOS/ml; at higher dilutions and in presence of ox serum t,hat had some antiviral activity the yield was greatly reduced. RESULTS

E’$ect of Ultraviolet Irradiation upon

Certain

Properties of NDV

Infectivity. The infectivity of the virus is destroyed by UV irradiation according to the kinetics of a single-hit curve. Under the conditions mentioned, 1 hit occurs every 12 secondsfor either strain of NDV suspended in PBS at pH 7.4. Hemagglutination. Virus irradiated with an average UV dose of 15 hits was found to possessthe same HA titer as that of a nonirradiated control sample. ,4n exposure t,o 150 hits reduced the HA capacity of the virus to 50 %. Adsorption. The ability of the UVI virus to adsorb onto monolayers was studied by measuring the HA titer in the supernatant inoculum after various periods of adsorption. Virus sampleswhich had received an average dose of 15 or 150 hits were compared with a nonirradiated cont,rol. It was found for both dosesthat the UVI virus adsorbed at a rate not detectably different from that of active virus. (Active XDV has an adsorpt,ion velocity constant of 5.0 to 7.1 X 10Pgcell-’ min? at 37” in PBS at pH 7.4.) The remaining hemagglutinin in the sample irradiated with 150 hits also adsorbs as well as does the active virus. Enzymatic activity. The enzymatic activity of UVI virus was determined by the latter’s ability to adsorb to and elute from chicken erythrocytes; 4 X IO8UVI particles (irradiated with 15 hits) were permitted to adsorb to IEBC for 30 minutes at 4”. The cells were then washed three times in the cold to remove free virus in the supernate. The virus-cell complexes were finally resuspended in the original volume of warmed PBS and incubated at 37” for 30 minutes to allow elution of the virus. The cells were then centrifuged down and the supernates assayed for their HA titers. The results showed that NDV irradiated with 15 hit’s

INTERFERENCE

WITH

NEWCASTLE

DISEASE

77

VIRUS

adsorbs to and elutes from erythrocytes as well as does active NDV. appears, therefore, that UVI-NDV retains its enzymatic activity destroy cellular receptors. All-or-None

It to

Effect of Interference

Preliminary experiments showed that interference consisted of an actual decrease in the fraction of yielders and that interference was of an all-or-none nature; either the cells become nonyielders (complete exclusion) or the cells release their normal yield of progeny virus at the normal rate (no interference). These conclusions are based on the following observations: Table 1 shows the results of a typical experiment in which cells were infected on the plate with 4 X lo* UVI particles, then superinfected with IO8 PFU 67 minutes after the first infection. Controls were run with UVI virus and active virus alone. The fraction of yielders was 43 % for the cells which received active virus alone and only 4% for the cells which had received UVI-NDV prior to their infection with active NDV. Figure 1 shows the release of new virus by the three types of infected cells described above. Curve A is a normal growth curve of NDV. Curve B represents the production of new virus in cells which escaped interference. The shapes of the two curves are nearly identical except for the different absolute values, i. e., 8.6 X lo3 and 8.6 X lo2 yielders for curve A and curve B respectively. A yield of 14 progeny particles per yielder is reached in both cases within 12 hours. It must be mentioned that this low yield, for the reasons pointed out under Methods, is a result of having only 2 X lo4 cells/ml in LA-YE plus ox serum. TABLE DECREASE

IN

THE

FRACTION

OF YIELDERS

1 SUBSEQUENT

T O INFECTION

WITH

UVl

VIRUS’= First infection with WI virus

4 x 108 4 x 108 none

Second infection with active virus

none 108 108

Yielders/ml

8.7 8.7

-10 x x

Total

102 103

2 x 2 x 2 x

cells/ml

10” 104 104

Fraction

of yielders

Negligible 4.3% f 0.5 43yc f 6.4

a Infection was carried out on the plate. The first inoculum consisted of 4 X lo* UVI particles in a volume of 1 ml. After an adsorption period of 30 minutes, the residual free virus was washed off and the layers were then superinfected with lo8 PFU. The time interval between the two infections was 67 minutes.

78

Time

in Hours

FIG. 1. All or none effect of exclusion. The curves are one after infection with active virus (A), UVI virus plus active virus only (C). In B, the active virus was added 67 minutes UVI virus. The time given starts at the moment the cells were active virus (about 2 hours prior to their resuspension in

IdentQication

of the UVI

Particle

as the Interfering

step growth curves virus (B) and UVl after infection with superinfected with nutrient medium).

Agent

The identification of the UVI particle as the interfering agent was shown unequivocally. The interfering activity was present only in [IVirradiated suspensionsof NDV, not in the virus-free supernates of such suspensions,nor in suspensionswhere the WI virus had been neutralized with specific anti-KDV serum. Moreover, as will be shown later, there was a direct correlation between the multiplicity of the CVI virus and the amount of interference obtained. Overcoming

of Interference

In the course of this work it was noticed that a fraction of cells would escaDeinterference no matter how many UVI particles had adsorbed per cell. The following results demonstrabed that these persisting infective centers consisted of actual yielders and not of free virus particles: (1)

IXTERFERENCE

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VIRUS

79

The persisting infective centers were destroyed by three successive freeaings and thawings, although this treatment did not inactivate free virus. (2) Anti-NDV serum which neutralizes free virus did not decrease the number of persisting infective centers. (3) The yield of new virus per yielder in this fraction was not significantly different from that of normal yielders. It was also possible’to show that this fraction was not made up of cells which had not’ been infected with UVI virus but only with the superinfecting active virus. In an experiment in which a suspension of siugle cells was infected with UVI virus at a multiplicity of infection of 200, 3 % of the cells still escaped exclusion when superinfected with active virus also at a multiplicity of 200. Moreover, this fraction of noninterfered cells was only observed when cells treated with UVI virus were superinfected with active virus. Cells which received UVI virus alone showed only one-tenth to one-eighth as many yielders, the latter arising from infection by survivors of the irradiated virus population. To elucidate the effect of the superinfecting virus, several experiments were done in which cells first infected with UVI virus were superinfected with varied multiplicities of active virus. The multiplicities of both agents were in all cases more than suflicient to infect all the cells. The data of Table 2 show that the fraction of cells which escaped interference increased with the multiplicity of the superinfecting virus. There exists therefore for each interfered cell a small probability that it can be superinfected by any given active virus particle. From the results obtained, either the maximum value of this probability is very small for all the cells, i.e., less than 4 %, or it has a value of 1 for a small fraction of the cells only. Whether this overcoming of interference results from a property characteristic of t,he virus or of the cell, remains to be determined. Number of UVI Particles

Required to Induce Interference

Let us assume that the virus particles (active or UVI) infect the cells in a random manner. The fraction of cells C(r)/C, infected by r particles is then given by the term of the Poisson distribution function: C(r) co where m is the multiplicity

m’e-” r. I

of infection

(1)

and Co the total number of cells.

80

BALUDA TABLE

IXCREASE

IN THE INCREASE

FRACTION OF CELLS IN THE MULTIPLICITY

2

WHICH ESCAI~ES INTERFERENCE WITH OF THE SUPERINFECTIXC VIRUS

Yielders/total

Experiment

1”

controld (no LJVI Experiment 2”

virus)

controln (no UVI Experiment 3

virus)

control”

virus)

(no UVI

9 45 90 9 9 45 90 9 9 18 90 180 9

3.670 12% 17% 44% 3.5% 6.5y0 9.1% 36% 4.2y0 7.9% 13.3% 15.3% 29.4%

f f f f f f f f f f f f f

cells

0.5 1.1 1.2 5.4 0.2 0.5 1.8 2.2 0.7 0.8 I.0 1.2 3.9

Yielders

fr;;ctml,of

AN

as

0.08 0.27 0.39 1.00 0.10 0.19 0.25 1 .OO 0.14 0.27 0.45 0.52 1.00

n In experiment 1, lung monolayers were infected with UVI virus at a multiplicity of infection of 9; 35 minutes later they were superinfected with active virus at the given multiplicities. h In experiment 2, the multiplicity of the UVI virus was 90 and 2 ml of antiNDV serum at a dilution of 1: 10 in PBS was added to each culture after the second infection. c In experiment 3, the multiplicity of the UVI virus was 90 and serum was again added after the second infection. Aliyuots from each test sample were frozen and thawed three times; this treatment eliminated 90% of the yielders. In addition the yield per yielder in each sample was not significantly different from that in the control. rl The control samples received only PBS instead of UVI virus.

If there is a critical number, say i + 1, of UVI particles required to produce exclusion, the fraction of cells, C,/C,, capable of becoming yielders by superinfecting active virus is given by the expression:

where m indicates the multiplicity of infection of the UVI virus. If a single UVI particle is sufficient, to induce exclusion, equation reduces to :

(2)

INTERFERENCE

WITH

Multiplicity .8

Virus

NEWCASTLE

of Infection 1.6 2.4 I

Concentration

DISEASE

VIRUS

81

3.2 I

(x 10s7)

FIG. 2. Determination of the multiplicities of infection as a function of the input concentration of active virus. Lung cells were infected on the plate with a volume of 1 ml of an inoculum containing varying concentration of active NDV. The inoculum was adsorbed for 30 minutes; the number of yielders was subsequently determined in the usual manner.

Similarly, if 2 UVI particles are required:

By plotting the experimental value of C,/C, as a function of m, it is possibleto determine for which value of i this curve fits equation (2) best. To make such a comparison, the multiplicity of infection of UVI virus must be known. To this purpose the multiplicity of infection of the active virus before irradiation was determined as a function of its concentration. Since it had been shown previously that adsorbability was not affected by the UV dose given (15 hits), the same dependence of the multiplicity of infection on the virus concentration was assumedfor the UVI virus. Figure 2 showsthe fraction of nonyielders as a function of the virus concentration in the inoculum and the corresponding calculated multiplicity of infection. The relationship between bhe interfering ability and the multiplicity

82

BALUDA

of the UVI virus was then determined. The general plan of the experiments was to infect a constant number of cells with varied amounts of UVI virus, and to determine the fraction of yielders. The dat,a are shown in Kg. 3, together with the theoretical curves corresponding to equations (3) and (4) shown above. As may be seen, the experimental results fit best the curve which requires t,hat’ only 1 WI particle be sufficient to produce interference. The data of Table 3 confirm in an independent manner the conclusion reached above. It was found that for a given concent,ration of UVI virus the multiplicity of exclusion is identical to the multiplicity of infection of active virus at the same concentrat,ion. Moreover, the plateaus giving 100% yielders and 100% interference are reached at the same concentration of act,ive and WI virus respectively.

;

ul.40= 0” m 2 f .20.c I c z” .lO.08 .06’

0

I I 1.0 2.0 Multiplicity

FIG. 3. Determination of the Curve A is a theoretical plot, plot of equation 4 CM/C, = emrn fraction of cells which became various multiplicities of infection

\, of

3.0 UVI

, \ 4.0 Virus

f 0

number of particles required to induce exclusion. of equation 3 (“JCO = e-” and curve B is a (1 + m). The experimental points represent, the yielders when superinfected wit,h act,ive virus at, of the WI virus (15 hits).

INTERFERENCE

WITH

NEWCASTLE

TABLE

Determination

Concentration

of virus

___~

2.5 5.0 2.5 5.0 2.5

x X x x x

109 IO8 108 107 107 Determination

of Multiplicity

Fraction

f f f f f

Fraction

of

yielders

2.5 X lo9 5.0 x 108 2.5 X lo* 5.0 x 10’ Control (No UVI virus)

of Infection

0.044 0.047 0.074 0.141

83

(Active Virus) Yielders, fraction of maximum

,047 ,058 .019 ,016 ,014

of Multiplicity

VIRI:S

3

of yielders

0.412 0.516 0.354 0.327 0.240

DISEASE

1.0 1.0 1.0 0.77 0.52 of Exclusion Fraction of yielders corrected for overcoming of interference

Multiplicity of infection

1.5 0.84

Yield per yielder

55 68 78 ” o

(UVI Virus)

Yielders, fraction of maximum

f f f f

,005 ,004 ,006 ,009

0 0 0 0.10

0 0 0 0.26

0.387 f

,046

0.387

1.0

Yield Multiplicity of exclusion

b D 1.35

Per

yielder

48 49 81 72 90

n Not determined. b The experiments were carried out with infection in suspension. Anti-NDV serum at a dilution of 1:lO in PBS was added to the cells for 15 minutes after the last infection. In the determination of the multiplicities of exclusion, a volume of 1 ml of superinfecting active virus containing 2.4 X lo9 PFU was added to thr cells 50 minutes after the infection with UVI virus.

Speed of the Interference Reaction Preliminary experiments had shown that exclusion was induced very rapidly. For instance, monolayers infected with UVI virus at a multiplicity of infection of 12, washed free of unadsorbed virus, then super-

infected 11% minutes later with active virus at a multiplicity of 12, showed exclusion in 93 % of the susceptible cells. In another case in which the multiplicities of the UVI virus and of the active virus were both 80, 28% of the cells showed exclusion when the interval between the two infections was only 1 minute. The great, rapidity with which interference took place called for more

81

BALUD.4

accurate means of determining the speed of the reaction t,han those described above. This was attempted by a study of the kinetics of infection of the cells. If we assume that the virus particles adsorb to host cells at random and that it takes 7 minutes for exclusion to be induced in a cell after the adsorption of one UVI particle, we arrive at the following expression for the fraction (1 - CJC,) of nonyielders when t,he 131 and the active particles are added to the cells simultaneously:

1+

vi ,”

vi + v, Pnr

exp - F (1 - eekCnT) :o

(5) *

where C,/C, is the fraction of yielders after a very long time of adsorption (considered to be infinite), vi and Va are the initial concentrations of UVI and active virus respectively, k is the adsorption velocity constant shown previously to have the same value for both active and UVI particles and COis the total number of cells in a monolayer. Theoretical values of (1 - C,/C,) were calculated from the above equation for values of T ranging from 0.1 to 5 minutes. These values are plotted on a semilogarithmic scale as a function of V, in Figs. 4 a and b for comparison with t,he experimental values. The experimental values of (1 - C,/C,) were determined under t’he conditions stipulated in deriving equation (5). Monolayers were infected with I ml of a given high concentration of UVI virus (Vi) and variable amounts of active virus (V,). The inoculum was adsorbed for 50 minutes (this time being considered infinite for all practical purposes) and the fraction of yielders C,/CO was determined. In one experiment the cells were treated with antiserum at the end of the adsorption period. Controls were run to determine the fraction of the cells in which interference is overcome in order to make t,he necessary corrections in the calculations. h comparison of the experimental findings with the theoretical curves shows that 7 falls between 0.2 and 1 .O minute with a most probable value of 0.4 to 0.5 minute when Vi is approximately equal to 2 X log UVI particles/ml (Fig. 4a). When vi is equal to 6 X log particles/ml the experimental data show a good fit to the curve constructed wit,h a theoretical value of 0.1 minute for 7 (Fig. 4b). The differences in the value of 7 suggested a dependence of 7 on the multiplicity of the UVI virus. That t,his dependence actually exists was * See appendix

for t,he derivation

of this

equation.

INTERFERENCE

WITH

NEWCASTLE

DISEASE

85

VIRUS

1.0

.8 .6 .4 20” I I

0.1 min

FIG. 4a and 4b. Determination curves of (1 - C,/C,) versus

Vafor

of 7. The different Vi

vi

+

v,

straight values

lines represent of 7 according

the theoretical to the equation:

e-(Va/Co)(l--e-~C~‘) e--kc”+

Figure 4a holds for Vi = 2 X lo9 UVI particles = 6 X IO9 UVI particles in the inoculum.

in the inoculum

and

Fig.

4b for

Vi

borne out by the following experiment in which the concentration of active virus was kept constant at 3.1 X 10’ PFU/ml, and the concentration of UVI virus was varied from 1.4 X IO* to 3.5 X lo7 particles/ml. The general experimental procedure was identical to that employed in

TABLE DEPENDENCE

.\lulti licity &virus

OF T ON

of

Yielders/total

3.2 1.6 0.8 (Control)

THE

10 f 13.5 f 18.5 f 22.5 f

none

4

MULTIPLICITY

cells

0.770 0.8% 3.0% 1.2%

OF THE

UVI

1-g (after correction for overcoming of interference)

0.65 0.49 0.24

VIRCW

T (minutes)

1.2 1.9 6.0

a The multiplicity of infection of the active virus was kept constant at 0.71 and the multiplicity of the UVI virus was varied as shown in the table. Both agents were added simultaneously to the cell layers and permitted to adsorb for 50 minutes. The fractions of yielders were then determined and 7 was calculated from equation (5) (10% of the control was taken as the fraction of cells in which interference is overcome).

the determination of 7. The results are shown in Table 4. It can be seen that 7 increases with decreasing multiplicity of the UVI virus. Addition

of UVI

Virus after the Active Virus

The growth curves shown in Fig. 5 indicate that the UVI virus did not influence significantly the production of new virus when added 72 minutes after active NDV (compare curve B with the control A and the interference control C). The data of Table 5 show that UVI virus did not decrease significantly the fraction of yielders when it was added as soon as 8 minutes after active KDV. Duration

01 Exclusion

No decrease in exclusion was found if active virus was added as late as 141 minutes after the interfering virus. Exclusion was followed over a longer t’ime interval by studying the release of new virus from intact monolayers. Figure 6 shows that cells which had received UVI virus 13 hours prior to the active virus (curve B) released even after 24 hours of incubation one-seventieth as much new virus as the control cells (curve A). In this experiment exclusion appears to occur to a greater extent than usually observed. The use of intact monolayers instead of single cell suspensions may account for this effect.

INTERFERENCE

WITH

5

NEWCASTLE

7 Time

9 in Hours

DISEASE

II

VIRUS

87

13

FIG. 5. Effect of adding UVI virus after the active one. Two lung monolayers were infected with active NDV at a multiplicity of infection of 160. A third plate received UVI virus at the same multiplicity. The inocula were adsorbed for 30 minutes, then LA-YE + 10% ox serum and 5% EE was added for an additional 30 minutes. Seventy-two (72) minutes after the first infection the layers were superinfected; one of the two layers previously infected with active virus received UVI virus at a multiplicity of infection of 160 (B); the other one received PBS only (A) ; the third plate was superinfected with active virus also at a multiplicity of 160 (C) The fraction of yielders in the normal control (A), the interference control (C) and the experimental plate (B) were respectively: 0.76, 0.10, and 0.57. Single step growth curves were run on the infected cells from A, B, and C in LA-YE plus 10% ox serum and 5% embryo extract. The time given starts at the moment, t,he cells were infect,ed with active virus.

The rate of appearance of necrotic areasin the cultures suggestedthat exclusion lasts between 26 and 48 hours. Whereas many necrotic areas had appeared in layers which received only active KDV, the cultures pretreated with UVI virus 13 hours previously were still in excellent morphological condition 26 hours after the second infection. However, 53 hours after the second infection many small necrotic foci had appeared in layers which had received both active virus and UVI virus. Although

88

BALUDA

TABLE LACK

OF

EFFECT

WHEN

UVI-NDV Interval infections,

(active

virus

AFTER

between minutes

alone) -36

2” Control Control

ADDED

36 16

I”

Control

5 IS

(interference rontrol) 8 8

(active (active

virus virus

alone) alone)

THE

ACTIVE

Fraction yielders,

of Y.

VIRUS Yield per yielder

47.5 37.0 43.0 4.5

f f f l

2.2 2.0 3.4 0.3

66 133 35 33

20.0 18.0 22.5 21.5

f f f f

2.2 2.0 2.6 2.0

220 306 271 265

u In experiment 1, the multiplicity of infection was 27 for both t,he UVI and active virus. b In experiment 2, the multiplicities were 54 and 108 for the active and t,he UVI virus respectively. c In both experiments the unadsorbed virus was tjhoroughly removed after each infertion. the

fewer in number, small necrotic areas had appeared also in cultures which had received only UVI virus. The cells which had been infected only with active NDV were completely destroyed at this time. Noninfectious

Hemagglutinin

Infective supernates from layers or cell suspensions in which exclusion occurred in approximately 90 % of the cells always showed PPU: HA ratios larger than 106. It can, therefore, be concluded that cells in which exclusion occurs do not produce noninfectious hemagglutinin. E.fect of Increasing

UV Dose upon th,e Interfering

Activity

of the Virus

Lung cultures were infected simultaneously with active NDV and with UVI virus which had received an average of 10, 50, or 100 hits. The multiplicities of infection of the UVI and active particles were 6.4 and 3.2 respectively. One lung culture received active virus only. The inoculum was adsorbed for 50 minutes, and the fraction of yielders in each culture then determined. Such experimental conditions permitted the determination of r as mentioned previously. The data of Table 6 show that NDV irradiated with 100 hits is as good an interfering agent as virus irradiated with 10 hits only. A value of 0.4 minute was found for 7 with all three UVI virus preparations.

INTERFERENCE

WITH

NEWCASTLE

DISEASE

VIRUS

89

IS-

em (

7-

6,

,

/

I 58

I2

I6 Time

20 in Hours

24

28

FIG. 6. Duration of exclusion. The interfering inoculum consisting of 4 X 109 UVI particles (1 ml) was adsorbed for 60 minutes. The unadsorbed virus was thoroughly removed; then 5 ml of LA-YE were added. The cell layer was superinfected 13 hours later with 4 X 109 active particles. Again the free virus was removed, then 5 ml of LA-YE were added. Samples from the supernate were assayed for new virus at various time intervals (curve B). Curve A shows the release of new virus by a control culture which received active virus only; Curve C represents the production of new virus by a culture that had been infected with UVI virus only; the virus is produced by the survivors of the UVI virus population. The time given starts at the moment the cells were superinfected with active virus.

Effect of Anti-NDV

Serum

The addition of anti-NDV serum to the lung cells after infection with UVI virus but before infection with fully active virus had an unexpected effect since it could remove interference under certain conditions. The data of Table 7 show the effect upon exclusion of the variation

90

BALUDA

TABLE OF UV

E:FFECT uv

dose

Fraction yielders 7 (min)

the

DOSE

ON

10 hits I of 1 0.264 +Z 0.016 / I 0.4

/

THE

6

INTERFERING

50 hits 0.283

f

0.016

Experiment number

1

Control Control

Time interval between WI virus and serum, minutes

ANTI-XDV

Fraction of yielders to total cells

30 60 90

18.2% 16.6% 16.5% 3.1% 33.070

experiments lung cultures for 10 minutes. After the anti-NDV serum for 15 were superinfected with the second infection the the excess free virus. The

0.016

Control UVI) 0.446 f

(no 0.021

employed

previously

in

7 WITH

19.50/, 20.0% 10.5% 1.3% 21.0%

(no serum) (no UVI)

u In both plicity of 32 treated with the cultures minutes after to neutralize

t,o that

13’/$ 28 44

(no serum) (no UVI) 2

Control Control

OF EXCLUSION

f

VIRUS”

0.4

identical

TABLE INHIBITION

0.242 I

was

OF THE

100 hits

0 .4

(L The experimental procedure determination of 7.

ABILITY

f f f f f -. f rt f f f

1.1 1.1 0.7 0.3 1.2 1.2 1.1 1.2 0.5 1.7

SERUW

Yield per yielder

27 61 36 6 38 _____36 49 92 31 105

Fraction yielders control yielders,

of to 5%

100 100 50 6 100 55 50 50 9.4 100

were infected with UVI virus at a multitime intervals given, these cultures were minutes. The antiserum was removed and active virus at a mult,iplicity of 32. Ten cultures were again t.reated with antiserum fractions of yielders were then determined.

in the time interval between infection with UVI virus and treatment with antiserum. Lung cultures were infected with GVI virus at a multiplicity of 32 for 10 minutes. After various time intervals, a volume of 2 ml of anti-NDV serum (1: 10 in PBS) was added to the layer. After 15 minutes t)he antiserum was removed by three washings with PBS, the cells were t,hen superinfected with active virus at a multiplicit,y of 32. Ten minutes after t,he second infection, the cells were again treat.ed with antiserum t,o neutralize the free virus. A control was run without adding

INTERFERENCE

WITH

NEWCASTLE

TABLE EFFIKT

OF ANTI-NDV 7) minutes

Fraction of yielders Yield per yielder

0.237 f0.013 62

SERUM 11 minutes

0.223 f0.016 83

UPON

DISEASE

91

VIRUS

8 NORMALLY

INFECTED

1% minutes

CELLS”

19) minutes

0.210 f0.016 73

0.275 f0.014 39

0.265 f0.012 44

-

u The inoculum contained 5 X lo9 PFU and was adsorbed for only 5 minutes. The layers were washed twice with PBS before adding anti-PIJDV serum (1:lO in PBS). The control received normal rabbit serum diluted 1: 10 in PBS. Freezing and thawing the infected cells three times in rapid succession destroyed 90% of the yielders.

antiserum after infection with the UVI virus. A second control did not receive UVI virus. The results show that anti-NDV serum added up to 28 minutes after UVI virus prevents the occurrence of exclusion. When the antiserum is added later-from 30 to 90 minutes-only 50% of the susceptible cells become yielders. In the light of these results the effect of anti-NDV serum upon normally infected cells was reinvestigated. The data of Table 8 show that anti-NDV serum added to cells as soon as 7>5 minutes after active NDV did not decrease significantly the number of yielders nor the yield of progeny virus. Through the effect of anti-KDV serum the first difference between UVI and fully active NDV had been detected in the sequence of events following adsorption of the virus particle to the lung cell. The interfering ability of the UVI particle remained sensitive to antibody indefinitely, at least in 50 % of the cells, whereas the infecting ability of the active particle could not be inhibited after 7 minutes at most. It appears, therefore, that exposure of NDV to UV irradiation renders this virus for at least 28 minutes, unable to penetrate into the host cell. In contrast, normal rabbit serum, RDE, and trypsin were found to be unable to inhibit exclusion when added to cells within 28 minutes after the addition of IJVI virus. These observations support the hypothesis that, the inhibition of exclusion by anti-NDV serum is brought about by the specific &ion of the antibody upon the UVI particle. This implies that the UVI part,icles remain at the cell surface and that exclusion takes place at the cell surface.

92 Fate of the Superinfecting

BALUDA

Virus

Although exclusion could be completely overcome when antiserum was added up to 28 minutes after the UVI virus, cells which were superinfected with active virus 941 minutes after the addition of the interfering agent and were then treated with anti-NDV serum 1935 minutes after the UVI virus infection did not yield any progeny virus. This experiment shows that the fate of the superinfecting virus is very different from that of a virus particle infecting a cell to which no UVI particle had attached. The superinfecting virus either became inactivated after its adsorption to the resistant cells or was prevented from penetrating the host cell thereby remaining susceptible to neutralization by specific antibodies. Additional information on this point was given by observations which did not involve the use of antiviral serum. It was found on one hand that there was on the average a twenty-fold drop in the amount of free active particles eluted from cells which had received UVI virus prior to the active virus if compared with similar supernates of cells infected with active virus only. It appears from this result that the superinfecting virus either was dest#royed or became unable to elute. Moreover, a cell infected by active virus under conditions in which the latter was excluded, did not yield new virus during an incubation period lasting for 6 days. Since exclusion eventually disappears within 60 hours after the infection of the cell with UVI virus, the superinfecting virus must have become inactivated. Thus all the available evidence concurs in showing that in this form of interference the excluded virus becomes inactivated, perhaps on the surface of the cell. Host Cell Response Exclusion of superinfecting virus was the only detectable effect of the UVI particles on the cells. An outstanding fact was the absence of visible morphological damage in the cells to which the UVI virus alone had adsorbed. These cells took up the vital stain, neutral red, as well as did normal cells. Moreover, when the progeny of the surviving fraction of the UVI virus population was kept negligible by adding antiserum to the growth medium, the lung layers remained intact for 7 days, their normal life span. On the contrary, cells infected by active virus can be clearly recognized as dead within 24 hours. However, the behavior of single cells infected with UVI virus should be investigated more thoroughly for their ability to produce clones.

INTERFERENCE

WITH

NEWCASTLE

DISEASE

VIRUS

93

DISCUSSION

The results of this study show that NDV rendered noninfective by ultraviolet radiation can exclude homologous active particles adsorbed to the samehost cell at a later time. The interfering activity is a property of the virus particle which is expressed upon its attachment to the cell. This property of NDV is resistant to large doses of UV radiation-in fact, it appears to be lost only when the UVI particle becomesincapable of adsorbing to the cell. It is not known whether the persistence of the enzymatic activity of the virus particle is required for the inducement of exclusion. However, some information on this point can be deduced from the fact that NDV heated at 56” for 1 hour does not interfere (Baluda, unpublished data). Heated virus has presumably lost its enzymatic activity but can still adsorb to cells. These findings suggest that adsorption to the host cell is not sufficient to bring about exclusion and that persistence of the enzymatic activity may be essential. UVI virus also losesits interfering ability upon heating. After the adsorption of the UVI virus, there is a maximal time interval during which the cell can still be superinfected by active virus. This time interval, which has a maximum of approximately 6 minutes when only 1 UVI particle has been adsorbed, decreases with an increase in the multiplicity of infection of the UVI virus. This multiplicity effect may be interpreted in two different ways if we picture exclusion to be the result of a spreading change at the surface of the cell, as suggestedfrom the fact that the ratio of the viral surface to the cellular surface is of the order of 1: 20,000. On one hand, this type of change may be brought about by a fast reaction triggered by the UVI virus coming into contact with certain cellular constituents. The spreading time would be short compared to the average time required to infect a cell with the UVI virus, even at the highest multiplicity of this virus. In this case,the multiplicity effect noted above may be interpreted as a reflection of the different time required to infect all the rells with the UVI virus. The minimal time interval required would represent the time necessary for 1 UVI particle to reach a triggering site. Since, with a multiplicity of 140, interference is induced in 0.1 minute or less, the interfering reaction is started either as soon as the IJVI particle is adsorbed or very shortly thereafter. On the other hand, the surface change may be caused by a slow reaction; the maximal time interval during which the cell can be superinfected would represent the time required for the reaction induced by 1 particle to spread over the entire cell surface. The multiplicity effect,

94

BALL-D.4

may now be interpreted as an increase in the number of foci where the spreading changes are initiated. Under either hypothesis such surface changes must be reversible in order to account for the finding that neutralization of the UVI virus removes exclusion. It was found that when anti-nTDVr serum is added t,o the cells 30 minutes or more after the UVI virus, interference would be removed in only 50 % of the cells. This finding shows that, an irreversible form of interference may arise because the previously reversible changes OF curring at t,he cell surface become “fixed” through some secondary react)ion. Very st’rikingly these changes occur almost simultaneously in all t,he affected cells; there is a rather sharp transition from reversibility t,o irreversibility within a few minutes. The finding that IJVI virus does not affect, the production of new virus when it is added to cells already infected with active virus is in agreement with t’he fact that IV1 virus affects the early st,epsof infection by active virus. APPENDIX

Derivation of the Equation tion by Active and ITI

Describing Virus

the Kinetics

of Simultaneous

Infee-

The number of virus particles 17adsorbed at any time t from a suspension containing originally V, particles is given by the equation: I’ = 17, (1 - e-w)

(1)

where k is bhe adsorption velocit’y constant, shown previously to have the same value (7.1 X 1O-g cell-r min-I) for both the active and t,he IJVI particles, C, is the total number of cells in a lung culture. From the Poisson probability function, t,he number of cells that’ have adsorbed virus at t,he time t is then: (y = (7, [I _ e-(l’olc,)(l-e-kc,r)] (2) and the number of cells dC that adsorb virus for the first t)ime during the imerval dt is:

Let us now assumet,hat it takes 7 minutes for exclusion to be induced in a (bellafter the adsorpt,ion of I-TJVI particle. Moreover, let us consider

INTERFERENCE

WITH

NEWCASTLE

DISEASE

VIRUS

95

the case in which the UVI and the active particles are added ximultaneously to the cells. Then, for 7 minutes, the infection of the host cells by the active virus will proceed unhindered by exclusion and at = r we will have C(T) yielders: C(,) = co [I _ e-(l,“/co)(l--e-kC,r)] (4) t

where V, is the initial concentration of active virus. For any time greater than 7 the number of cells, C; exclusion has been induced is: C&

>

7)

=

Co1

1

_

,-c~i/c,,[l--e-kCu(t~r)l

(t

> T), where

1 (3

where Vi is the initial concentration of UVI virus. The number of cells still infectable is then: 1 _

cl&

>

T)

=

COe-‘vilco’L’-“-kCO(t--r)l

and from equations (3) and (6) the number adsorbing active virus during becomes:

(f3

of cells still infectable

and

dt

dC _ dt

=

~CoV,

The total number

e--kc,te-(

of yielders

v,/CO)+-

wet

e -(V,/(~“)[l-s-kC,(l~r)]

after a very long t’ime

C, = C(7) + Jm kCoV, e-~c"~e-~~'"/co~~l~e-kCo"'e-'Vi"O'll-"-~~"~~--r~l

(t

(7) = 00) is then: dt

(8)

7

After integration

and replacing

Since the experimental

C(7) by its value given in equation

T/a + vi values of c will always

(4) :

be very large, i.e.

greater than 100, the second term on the right-hand side of equation (9) is negligible. After a final rearrangement of the terms of equation (9), the total fraction of nonyielders (1 - CJC,) is given by the following expression :

(10)

96

BALUDA

ACKNOWLEDGMENTS The author Dulbecco and

acknowledges with pleasure the guidance of Doctor Harry Rubin during the pursuit

of Professor Renato of this problem.

REFERENCES F. B. (1948). Studies on Newcastle Disease Virus. I. An evaluation of the method of titration. J. Esptl. Med. 88, 233-240. DLJLBX~~, R., VOGT, M., and STRICKLAND, A. G. R. (1956). A study of the basic aspects of neutralization of two animal viruses, Western Equine Encephalitis virus and Poliomyelitis virus. Virology 2, 162-205. FAZEKAS DE ST. GROTH, S., and EDNEY, M. (1952). Quantitative aspects of influenza virus multiplication. II. Heterologous interference. J. Immunol. 69, 16(t168. FAZEKAS DE ST. GROTH, S., ISAACS, A., and EDNEY, M. (1952). Multiplication of influenza virus under conditions of interference. Nature 170, 573. FRANKLIS, R. M., RUBIN, H., and DAVIS, C. A. (1956). The production, purification and propert,ies of NDV labeled with radioactive phosphorus. Virolog?y 3, 96114. GRANOFF, A. (1955). Noninfectious forms of Newcastle disease and influenza viruses. lrirology 1, 516-532. HANSON, R. P., UPTON, E., BRANDLY, C. A., .~ND WINSLOW, N. S. (1949). Heat stability of hemagglutinin of various strains of Newcastle disease virus. Proc. Sot. Exptl. Biol. Med. 70, 283-28i. HENLE, W. (1950). Interference phenomena between animal viruses: a review. J. Immunol. 64, 203-236. HENLE, W., and HENLE, G. (1944). Interference between inactive and active viruses of influenza. II. Factors influencing the phenomenon. Am. J. Med. Sci. 207, 717-733. HENLE, W., HBNLE, G., and ROSENBERG, E. B. (1947). The demonstration of one-step growth curves of influenza viruses through the blocking efl’ect of irradiated virus on further infection. J. Exptl. Med. 86, 423-437. HORSFALL, F. I,., JR. (1954). On the reproduction of influenza virus. Quantitative studies with procedures which enumerate infective and hemagglutinating virus particles. J. Erptl. Med. 100, 135-162. SALK, J. E. (1944). A simplified procedure for titrating hemagglutinating capacity of influenza virus and the corresponding antibody. J. Immunol. 49, 87-98. ZIEGLER, J. E., LAVIN, G. I., and HORSFALL, F. L. (1944). Interference between the influenza viruses. II. The effect of virus rendered noninfective by ultraviolet radiation upon the multiplication of influenza viruses in the chick embryo. J. Exptl. Med. 79, 379-400. BANG,