Factors Affecting the Sensitivity of Different Viruses to Interferon

Factors Affecting the

Sensitivity of Different Viruses

to

Interferon

JULES V. HALLUM, HARSHAD R. THACORE, AND JULIUS S. YOUNGNER

Departmentof Microbiology, School of Medicinle, UnuiversitY of Pittsburgh, Pittsbi,rgh, Pennsylvania 15213

Received forpuLblication8 May 1970

When the sensitivities tointerferon ofNewcastle disease virus (NDV) and

vesicu-lar stomatitis virus (VSV) werecompared by theplaquereductionmethod inchick

embryo cell cultures, NDVwasfound to be45-fold moreresistant than VSV. This

difference was exaggerated whena multiple-cycle yield inhibition method was

em-ployed. In markedcontrast,when thesamevirusesweretestedbyasingle-cycleyield

inhibition method, the difference in sensitivity tointerferonof the twoviruseswas

virtually eliminated. Further investigation showed that, in chick embryo cells

ex-posed to interferon, the resistancetoNDVdecayedmore rapidly than resistanceto

VSV. This findingexplained the divergent results obtained with thetwoviruses when

single-ormultiple-cycle replicationtechniques wereemployed. Experiments carried

outwith L cellsshowed that cellularantiviral resistance decayed muchmoreslowly

inthese cells than in chick embryo cells. Consequently, when measuredbytheplaque

reduction method in Lcells, no differencewas observed in the sensitivityto

inter-feron of VSV and

NDVpi,

a mutant of NDVwhich replicatesefficiently in Lcells.

Aprocedureis suggested for determining the relative sensitivitiestointerferon of

dif-ferent viruses under conditions which minimize the role ofdecay ofantiviral

resis-tancein the host cells.

Wide variations in the response of different

viruses to inhibition by interferon (3, 9-11, 13,

17) have suggestedaranking ofviruses based on

their sensitivity to this inhibitor (15). The

arbo-virusgroup is usually considered most sensitive;

vesicular stomatitis virus (VSV), vaccinia, and

other poxviruses are of intermediate sensitivity;

and Newcastledisease virus (NDV), herpes

sim-plex, and cytomegalovirus (9) arethe least

sensi-tive. An example ofthe basis forthis ranking is

the difference in sensitivity of VSV and NDV to

interferon when tested by a plaque reduction

method in chick embryo (CE) cell cultures; a

concentration of interferon which completely inhibits plaque formation by VSV has little or no

effect on NDV. Any mechanism which is

in-voked to explain the action of interferon must

takeinto account such variations in the

suscepti-bilityof different viruses. Theexperiments reported

inthis paper were designed to answer the

follow-ing questions. (i) Isthe variation in sensitivity of

viruses tointerferon in a given host cell a function

of themethod ofassay? (ii) What determines the

sensitivityofagivenvirus when tested in different

hostcellsby the same method of assay?

MATERIALS AND METHODS

Cell cultures. MouseLcells(clone 929) and primary

CEcell cultures were propagated and maintained in

Eagle's minimal essentialmediumplus 4% calfserum

as described in detail elsewhere (18).

Viruses. The Hertsstrainof Newcastle disease virus (NDVO)and asmallplaquemutant(NDVpi,clone 2) isolated fromLcells persistently infected with NDVo

were used in this study. The viruses were grown in CEcellcultures and in chick embryos as previously

described (14). Both NDVo and NDVpi replicatein

CEcell cultureswithout the productionofdetectable

amountsofinterferon.Infection ofLcells with NDVo results inanabortiveinfection whichyieldsfew infec-tive virus particles. In contrast, NDVpi replicates

efficiently in L cells and produces infective virus.

BothNDVoandNDVpiinduce interferonsynthesisin

Lcells (14).

The Indiana strain of VSV was propagated in L

cellsasdescribed earlier (18).

Preparation of interferon. L cell interferon was prepared as follows: L cells grown in 32-oz bottles (ca. 900 ml) were infected with NDVO at an input

multiplicityofinfection (MOI) of100plaque-forming

units (PFU) per cell. After 60 minofadsorption at

37C, the cellswerewashed, refed with medium, and incubated at 37C for48 hr. Theculturefluids were

harvested and clarified by centrifugation, and the

infectivitywasinactivated bytreatment atpH2for2

weeks.Interferonpreparationswerestoredat -20 C.

Chicken interferon was prepared in embryonated eggsby the method of Wagner(16)withthe WS strain

of influenza virus; details oftheprocedureshavebeen reported earlier (4).

Interferon assay. Plaque reduction assays were

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carried out in L or CE cell cultures as described

previously (18).

Twotypes of yieldreduction methodswereutilized.

(i) Single-cycle yield reduction was determined as

follows.Cellcultures wereexposedto serialdilutions

of interferonfor 20 hr at 37 C. Thecellswere washed

twice with growth medium and infected with the

appropriatevirus at an input MOI ofI. After

adsorp-tion for 60 min at 37 C, the cultures were washed twice

toremoveunadsorbedvirus, refed withmedium, and

reincubatedat 37 C.Fluids frominfected cultures were

harvested after the time required for a single cycle of

replication.In CEcellculturesthistime was 6 hr for

VSV and 10 hrfor NDVOand

NDVpi.

The per cent

inhibition of yield by interferon was calculated by

comparing the virus yield from cultures treated with interferon to the yield from untreated control cul-tures. (ii) Multiple-cycle yield reduction was

deter-minedin asimilar mannerexceptthat thefluidsfrom

infectedcellswereharvested 24 hrafterinfection, at which time the cells werecompletely destroyed.

RESULTS

nhibition

ofNDVandVSV byinterferonin CE

cell cultures: plaquereduction method. An

experi-ment was carried out to confirm that NDV is

more resistant to inhibition than VSV when

tested by the plaque reductionmethod in CE cell cultures.Serial0.5

log1o

dilutions of chick

inter-feron were added to CE cell cultures; after 20

hr at 37C, the interferon was removed, the cells

were washed, and the cultures were divided into

three groups. Eachgroupwaschallenged with40

to 60 PFU of either

NDVo,

NDVpi,

or VSV.

The per cent reduction ofplaques at each

inter-feron dilutionwascalculated from thenumber of

plaques of each virus in control cultures which

had been incubated with medium only. The

relativesensitivities of the three viruses to

inhibi-tionbyinterferonareshowninFig. 1. The

inhibi-tion of VSV

plaques

was used to calculate the

number of units of interferon present in each dilution, asshown in theabscissa ofFig. 1; one

unit of interferon was the amount required to

inhibit VSV plaque formation by 50%. The highest concentration of interferon (70 units)

used in this experiment produced only a 40%

inhibition of

plaque

formation bythe wild type

NDVO. In the case ofthe mutant

NDVpi,

15

units of interferon was required to produce a

50% plaque reduction. It is interesting that by extrapolatingfromtheinhibition data plottedin

Fig. 1, the titer of the chick interferon would be

15units,45units,and 700unitsper 3 ml ifNDVO,

NDVpi

, or VSV, respectively, was used as the

challenge virus. These data amply support the

claim thatNDVisrelatively more resistantthan

VSV to inhibition by interferon when tested by

theplaquereduction method in CE cell cultures.

Additional

experiments

were carried out in CE

cell cultures

by using yield

reduction methods in 100

90

-so8

z

0

5

o

s

z

z

w

C.

ccl 70F

60[

50

1-40_ 30 _ 20 _ 10 F 0

0.02 0.07 0.2 0.7 2.2 7 22 70 INTERFERON CONCENTRATION (UNITS/ml)

FIG.1. InhibitionofND Vand VSVby interferonin

CEcell cultures:plaquereduction method.Cellcultures

wereincubatedwith0.5logio dilutionsof interferonfor

20 hrat 37C, washed, andchallengedwith 40 to60

PFUofND V (0),ND

Vpi

(A),orVSV(0).Cultures

incubatedwithmediumalonewereusedascontrol. The

units ofinterferon shown on the abscissa were

calcu-latedfrom theamountrequiredtoinhibit VSVplaque

formationby50%(one unit).

order to

determine

the influence of

single

and

multiple

replication cycles

onthe relative sensitivi-ties of different viruses tochick interferon.

Inhibition

ofNDVandVSVby interferoninCE

cell cultures:

yield

reductionmethods.CE cell

cul-tures were incubated

overnight

with interferon

dilutions

and infected with either NDVO,

NDVp

i, orVSVat an

input

MOIof 1 asdescribed above.

After 24hr of

incubation,

when

complete

destruc-tion ofthe cellshad

occurred,

the culture fluids

wereharvested and

assayed

for

infectivity

in CE

cellmonolayers. Virus

yield

wasalsomeasuredin

control cultures

exposed

for 24 hr to medium only. A

plaque

reduction assay with the three viruses was carried out

simultaneously

in

inter-feron-treated

and control cultures to

provide

a

standard for

comparison

and to calculate

(from

theVSV

data)

thenumber ofinterferon units in each dilution used to treatthe cells. The results

plotted

in

Fig.

2show that

NDVo,

NDVpi,

and

VSV were lesssensitivetointerferon whentested

by the

multiple-cycle

yield

reduction methodthan

when tested

by

the

plaque

reduction

technique,

although the three viruses maintained the same

relative

sensitivity

to interferon. In the case of

NDVOX 500unitsof interferon

(as

determined

by

VSVplaque

reduction)

produced

a 35% decrease

VOL.

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[image:2.492.250.447.64.298.2]

100 90 so 70

z

0

x 50 z

0 40

0.30

20 10

0.007 0.03 0.12 0.48 1.9 7.8 31 125 500 INTERFERON CONCENTRATION (UNITS/3m1)

FIG. 2. Inhibition ofND Vbyinterferon in CEcell

cultures:multiple-cycle yieldmethod.Cellcultureswere incubatedwith serialfourfolddilutionsof interferon in

theusualmanner andchallengedwithNDVO,NDV,,j,or

VSVataninputMO! of Iasdescribedinthetext. The culturefluidwasharvestedafter24hr andassayed for infectivityinCE cellmonolayers. Culturesnot treated

withinterferonwereusedascontrol. Aplaquereduction assaywasalso carriedoutsimultaneouslyasdescribed

inthelegend of Fig. 1. Theyield(PFU/ml) ofprogeny virus harvestedfromcontrolcultureswas2.4 X 108for VSV,3.4 X 107 for NDVo, and 1.2 X 107 for NDVpi. Symbols: plaquereduction(brokenline), yieldreduction (solid line); forNDVO (Ol or U), NDV,,i (A or A),

and VSV(Oor @).

inplaque numbers butno inhibition of

multiple-cycle yield. With

NDVp

i, 500units of interferon

reduced plaque formation and multiple-cycle

yield by 72% and 20%, respectively; the same

amount of interferon produced complete

inhibi-tion of plaque formation and multiple-cycle

yield by VSV.

The relative sensitivities ofthe threeviruses to

inhibition by interferon was also tested by the

single-cycle yield reduction method. CE cell

cul-tures were exposed to interferon and then

challenged with the three viruses, as described

above. The culture fluid was harvested after the

time required for one cycle ofreplication, i.e.,6

hrfor VSV and 10 hr forNDVOand

NDVp,

.The

percent inhibition ofyieldofeach virusby

inter-feron is showninFig. 3. These experiments point

out that, when tested by inhibition of a single

cycle ofreplication,allthe viruseswereatleastas

sensitivetointerferonasthecomparisonstandard, 100

90 _

soF

z 0

I-a i z

I--Z hi

a:

hi

A. 70I 60 _

50

_---40 _ 30 _ 20

F

lo 0

0.007 0.02 0.07 0.22 0.7 2.2 7 22 70

INTERFERON CONCENTRATION (UNITS/mI)

FIG. 3. InhibitionofNDVandVSVbyinterferonin CE cell cultures: single-cycle yield reduction method. Cell cultureswereexposedtoiniterferonandchallenged withND VO (U),NDVpi(A),orVSV(0) atan input MOIof Iasdescribedinthe text. Theculturefluidwas

harvestedat 6 hrfor VSV and 10 hrforNDV. and

NDV,,i. Culturesnot treated withinterferonwere used

ascontrol. AplaquereductiontestusingVSV(0) was carriedoutsimultaneously forthedetermination of

in-terferon concentrations used in the experiment. The

yield (PFU/ml) fromcontrol cultureswas8.0 X106for NDVO, 3.7X 106forND

V,pi,

and 6.1 X107for VSV.

VSV plaque reduction. This finding is in striking

contrast to the wide variations noted in the

sensitivityofthedifferentviruses when testedby

the plaque reduction methodinCE cell cultures

(Fig. 1). It isdifficult, in the light of these results,

to designate NDVO a "resistant" virus since 1

unitofinterferon,determinedbytheVSVplaque

reduction data, reduced single-cycle yield by

50%.

Inhibition of

NDVpi

and VSV by interferon in

Lcellcultures. In view of thevariations in

sensitiv-ity ofNDV and VSV to interferon in CE cell

cultures, experiments were carried out to study

the inhibition ofthese agents by interferon in L

cells. By using the plaque reduction method, the

sensitivity of

NDVpi

and VSVto interferon was

determined inthe usualmanner. NDVQ was not

included in thisexperiment since it doesnot

pro-duce plaques inL cells. Figure 4 shows that, by

theplaque reduction method in L cells,

NDVpi

wasslightlymoresensitive thanVSVtoinhibition

by interferon. This is in sharp contrast to the

results in CE cell cultures (Fig. 1); in CEcells,

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VIRAL

100

90

-80j

z 0

0

co

x z

I.-z hi C.

70

-60

50

-40

30

201-Io

0

0.028 0.11 OAS 1.8 7.2 29 115

INTERFERON CONCENTRATION (UNITS/ml) FIG. 4. InhibitionofNDVpianzd VSVby interferon in L cell cultures. Cell cultures were incubated with variousconcentrations ofinterferonandchallengedwith 40to60 PFUofNDVpi (A)orVSV(0)asdescribed inthelegend ofFig. 1.

NDVpi

was found to be 15 times more resistant

tointerferonthan VSV. These results indicate that

the relative resistance ofa virus tointerferon, as

measured by the plaque reduction method, is

determinedinlargepartbythe hostcellinwhich

thetestsareperformed.Stewart and hiscolleagues

(13), who studied the relative sensitivitiesoffive

differentviruses in five different host cellsystems,

cametothe sameconclusion.

Single-cycle yield reduction tests were also

carriedoutin Lcellswith

NDVp

iandVSVasthe

challenge viruses. The results of these tests

showed that

NDVpi

was slightly more sensitive

than VSVtoinhibition by interferon, aresultnot

unexpected considering thedata obtainedby the

plaque reduction method. An explanation was

sought for the marked effect of the host cell on

the sensitivityofNDVandVSVtoinhibition by

interferon when measured by the plaque

reduc-tionmethod, atechniquewhich involves multiple

cycles of viral replication over a period of 3

days. Based on reports by several investigators

(1, 2, 7,16),itseemedpossiblethat the resistance

conferred uponCE cells bychick interferonwas

gradually lost during this time, allowing virus

replication and plaque formation, whereas the

resistanceconferredupon Lcells bymouse

inter-feronwas stable over the

period

ofdays required forplaque formation. Totestthis possibility, the

following experiment

was carried out.

Resitance

of CEandL

cell

culturesto

challenge

with VSV at different times after removal of

homologous interferon: plaque reduction method.

Serial twofold dilutions of chickand L

cell

inter-feronswereprepared, and each dilutionwasadded

to a large number ofhomologous

cell

cultures.

Control cultures received medium only. After

overnight incubation at 37 C, the interferon

dilutions

and the medium were removed,

re-placedwith 3 mlofmedium, and incubationwas

continuedat 37 C.Atthe time that theinterferon

dilutions wereremoved

from

the cultures and at

4, 8,

12,

16,and24hraftertheir replacementwith

medium, a dilution series of each

cell

type was

challenged

with40 to 60 PFUofVSV in the usual

manner. Bythis procedure, the plaque reduction

titer of the interferonwasdeterminedatdifferent times afterremovalofthe inhibitorfrom the cells, andtherateofloss of cellular resistancecould be

measured. Thedata inTable 1 show that

cellular

resistance was lost more rapidly in CE

cell

cul-turesthan in L

cells.

The experiments were

ter-minated

24 hr after the interferon

dilutions

were

removed since preliminary experiments showed that, when testedby the plaque reduction method, CEcell cultures

completely

lostresistancetoVSV by 48 hr after removal of interferon, whereas L

cells

retainedmostof their resistanceatthistime.

Resistance

ofCE cell cultures to

challenge

with

NDVO

orVSV at different

times

after removal of

interferon: single-cycle yield reduction method. CE cell cultureswere incubated

overnight

at 37 C with 1,000 units of chick interferon or with medium alone. Thefluidswereremoved, thecells were washed twice with medium, 3 ml offresh

medium

was added, and the cultureswere again incubatedat37 C. Ontheday the interferonwas

removed, and on each successive day, cultures were

challenged

with VSV orNDVO at aninput MOI of 10. Fluids from infected

cultures

were

harvested after one cycle of

replication

and assayed forinfectivity inCE cell monolayers. A

striking differencewasfound in therateoflossof

resistance when measured

by

using different

challenge

viruses (Fig. 5). In the case of VSV

challenge,

CE

cells

maintained their resistantstate

almost

unchanged

for the

3-day period

of the

experiment.Ontheother

hand,

therewasa

rapid

loss of resistance to

NDVo. Twenty-four

hours

after removal of the 1,000 units of

interferon,

single

cycle

yields

of NDVo were inhibited

71%

instead of the 96% inhibition measured

im-mediately after interferon wasremoved fromthe

cultures. Two

days

after removal of

interferon,

NDVP;

Ivsv

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TABLE 1.Resistanice of chickembryo(CE)andL cells tochallentgewith vesicularstomatitisvirus atdifferent

timesafter removal ofhonmologous interferona

Plaque-inhibitiontiter ofinterferonat

Cultures _ _ _ _

0hr 4hr 8hir 12hr 16 hr 24hr

CE cells 1,4I00 1,100 1,400 900 520 110

(100)b (78) (100) (64) (37) (8)

Lcells 12,000 10,000 11,000 11,000 7,200 8,500

(100) (83) (92) (92) (60) (71)

aCE or Lcell cultures were incubated overnight at 37 C with twofold dilutions of the appropriate

interferon preparation. The interferon dilutions were removed and replaced with 3 ml of medium,

and incubation was continued. Groups of cultures were challenged with 50 PFU of VSV at different

timesafter the interferon dilutions were removed.

bNumbers in parentheses represent the per cent of titer at 0

lhr.

only a 13

%/O

inhibition of yield was recorded; at 3

days the interferon-treated cultures were as

sus-ceptible to NDVO asthe untreated controls.

Effect ofconcentration of interferon on

single-cycle yields of VSV orNDVO in CE cell cultures.

Theexperiments which have been described show

that theapparent "sensitivity"ofagiven virusto

interferon depends on such factors as the virus

mutant, the host cell employed, and the type of

assay performed. The following experiment was

carried out in an attempt to minimize the

influ-enceofloss of resistance indefiningthesensitivity

ofNDVOandVSV to interferon and toestablish

a realistic basis for expressing the relative

sen-sitivities ofthese viruses.

CE cell cultures wereexposed to various

con-centrations of interferon and challenged with

NDVO or VSV at aninputMOI of 1, inthe usual

manner. Culture fluids were harvested after one

cycle ofreplication andassayed forinfectivity in

CEcellmonolayers.The inhibition ofsingle cycle yields of NDVo andVSVisplottedinFig.6. The

maximum level of inhibition ofyieldofboth VSV

and

NDVo

was produced with about 200 units

of interferon per culture; higher concentrations

of interferon did notfurther decrease theyields.

However, there wasasignificant differencein the

breakpoint of the inhibition curves ofthe two

viruses.Inthecase ofVSV, the maximum

inhibi-tion ofyieldwas4.0logs,whereas with NDVO the

maximuminhibitionwas 1.7 logs. This

charac-teristic difference in maximum inhibition of

NDVo and VSV wasexperimentally

reproduci-bleandprobably is an accurate measure of their

relativesensitivitiestointerferon.Experimentswere

performed which ruled out thepossibility(i)that the

NDVo population

contained

interferon-resistant variants and

(ii)

thatalarge population

of

reversibly

adsorbedNDVO

particles

waseluted. Io o

\ vsv

90 80

60 7 1:

z

5 \

z

w

40-w

a.

30 2 0

10 - NDVO

0 --L

-O 2 3

DAYS AFTER REMOVAL OF INTERFERO4

FIG. 5. Resistance of CE cell cultures to challenge

withNDV0 orVSV atdifferent timesafter removal of interferon:single-cycle yield reduction method. CEcell

cultures were incubatedovernight at 37 C with 1,000 unitsofinterferonorwith mediumalone.Cultureswere washed, refed with 3 ml ofmedium, andreincubated at 37C. At various timesafterremovalofinterferon, the

cultureswerechallengedintheusualmannterwitheither VS V(0)orNDV.(0)atanlinput MOI of10.Culture

fluid washarvestedafterone cycle of replication anid

assayedfor infectivityinCE cellmonolayers. Theyield

(PFU/ml) ofprogeny virusfrom control (untreated)

cultures was asfollows: day 0, VSV = 105,NDV. =

1.05 X 107;day 1, VSV = 106,NDVo = 2.1 X 107;

day 2, VSV = 1.6 X 10',ND Vo = 2.4 X 107; day 3,

VSV=5.0 X 106,NDVo =1.8 X 107.

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w

0

z

0

0

0 3.V

w

a 0.

o

0

.0

0 200 400 600 600 1000 1200 1400 1600

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INTERFERONCONCENTRATION (UN ITS/Dm1

FIG. 6. Effiect of concentration of interferon on single-cycle yields of ND V0 or VSV in CE cell cultures.

Cell cultures were incubated overnight at 37 C with

various concentrations ofinterferon and challenged in

the usual manner with either VSV (0) orNDVO (*) at an input MOlof 1. Cell cultures without interferon were used as controls. Culture fluid washarvested after

one cycle of replication and assayed for infectivity in

CE cell monolayers. The yield (PFU/ml) of progeny

virus from control cultures was 7.4 X 106 for NDVo and3.0X107forVSV.

In the latter experiment, anti-NDVserumn(18) was employed to neutralize all reversibly adsorbed

virus prior to virus replication.

DISCUSSION

The experiments we have described show that

thesensitivity of different viruses to inhibition by

interferon in a given host cell system depends on

the method of assay employed.For example, large

differences in the sensitivities of NDV and VSV

to interferon in CE cells were observed when

methods were used which permitted multiple

cycles of virus replication to occur. When test

methods were employed which involved only

single cycles of virusreplication, these differences

in sensitivity to interferon were obliterated. The

major difference between single- and

multiple-cycle replication methods is the elapsed time

between infection and the harvest of progeny

viruses. It is apparent from our data that the

anti-viral resistance established in CE cell cultures

in-cubated with interferon decreased when the

inter-feron was removed from the cells. The rate of

decrease of antiviral resistance was different for

NDVand for VSV. The more rapid disappearance

ofcellular anti-NDV activitywith time furnishes anexplanationforthe observationthat this virus

is apparently "resistant" to inhibition by

inter-feronwhen tested by the plaque or multiple-cycle

reduction methods. On the other hand, when

testedbythesingle-cycleyield reductionmethod,

whichminimizesthe decay ofestablished antiviral

activity, NDV appears to be as "sensitive" to inhibition by interferon as VSV, a virus considered highly sensitive to interferon.

Ourexperiments alsoidentifyoneof thefactors

which determine variationsinsensitivityof agiven

virus when tested in different host cells by the samemethodof assay. Whentestedbythe plaque

reduction method in CE and L cell cultures,

NDVpi

and VSV differ greatly in their relative

sensitivitytointerferon.In CEcells,15times more

interferon is required to inhibit

NDVpi

than to

inhibitVSV. In contrast, in Lcell culturesthere is

no significant difference in the inhibition of

NDVpi

and VSV by interferon when tested by

the plaque reduction method. Again, the factor which determines the "sensitivity" of a given

virusto interferon is therateofdecayof

cellular

resistance when interferon is removed; decay of

resistanceismuch morerapid in CE cells thanin Lcells.

Thevariationsin rate of

decay

ofcellular anti-viral resistance when interferon is removed may

explain

the observations ofStewart et al.

(13).

These workersfound

significant

differences inthe

sensitivity offive viruses to inhibition by inter-feron in cells from five different species. Since they employed the

plaque

reduction method,

conditions were provided which allowed rate of

decay ofcellular resistancetoplayamajor role in their

experiments.

Basedonourexperiences with

CE and L

cells,

it seems reasonable to suggest

that wide variations in the rateof

decay

of

resist-ancewould be encounteredin the five species of

cells

which

they

employed.

The

finding that,

in CE cell cultures treated

with interferon, resistance to NDV and VSV

waned at different rates aftertheremoval of

inter-feronwas

unexpected.

Two alternate

hypotheses

can be advanced to explain the

finding

that CE

cells treated with interferoncan remainresistant

to VSV at a time when they havebecome

com-pletelysusceptibletoNDVO .The first

hypothesis

isquantitativeandsuggests thatagreaternumber

of a single species of intracellular resistance

factor induced by interferon is neededto inhibit

NDVthan toinhibit VSV.

Initially,

after

incuba-tionwithinterferon,there isasufficient numberof

these factors to inhibit both viruses. As

decay

proceeds, the number of resistance factors per

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cell would decrease to a level which, though still greatenoughtoinhibit VSV,nolongeris sufficient

toblockreplicationofNDV. The second

hypoth-esis is qualitative and suggests that more than

one species of intracellular resistance factor is

induced by interferon. These different speciesvary

in their ability to inhibit different viruses and

decay at different rates. Thus, if two resistance

factors were induced in CE cells by interferon,

one factor active against VSV and the other

aganst NDV (the latter less stable than the

former), the results described inFig. 5 would be

obtained. Cell cultures would remain resistantto

VSV atatime whentheyhadbecomecompletely

susceptibletoNDV. In support of thequalitative

hypothesis, one can cite reports that interferon

preparationsoften containtwoormoremolecular

speciesof inhibitor (5, 6, 8, 12). Itseemspossible

that each of these molecularspeciescould induce

a different intracellular resistance factor. At

present,ourdata donotpermit a choice between

thequantitativeandqualitative hypotheses.

The closest we can come to the definition or

expression of the differences in sensitivity of

virusestointerferon is thefollowing.By usingthe

single cycle yield reduction method (which

minimizes the role ofdecay ofintracellular

re-sistancefactors) andby testing concentrations of

interferonwhichproducemaximum inhibitionof

yield, a "sensitivity factor" can be calculated.

This factor foragivenvirus(NDVO) isdefinedas

the ratio of the log10 dropin yield compared to

thelogio drop inyield ofa standard,

interferon-sensitive virus suchasVSV. Intheparticularcase

shown in Fig. 6, this "sensitivity factor" for

NDVo (2.35) was calculated as follows:

(maxi-mumlogio drop for

NDV./maximum

log10 drop

for VSV) = (4.0/1.7) = 2.35. Considering the

number of variables which influence the inhibition of viruses by interferon, the method suggested

for calculating the "sensitivity factor" may

permit a more rational ranking of viruses in

regard to their sensitivity to interferon than has

been available previously.

ACKNOWLEDGMENTS

The excellentassistance of MarionKelly andNancy Kidder is gratefullyacknowledged.

Thisinvestigationwassupported byPublic Health Service

re-searchgrantAI-06264 from theNational Institute ofAllergyand Infectious Diseases.

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