Interference Among Group A Arboviruses

(1)

Interference

Among Group A

Arboviruses

EUGENE ZEBOVITZ AND ARTHUR BROWN

BiologicalSciences Laboratories, Fort Detrick, Frederick, Maryland 21701

Received for

publication

29 July 1968

Interference among group

A

arboviruses

is

described

which

does

not involve the

mediation

of

interferon. Interference was observed

only if the

interfering

virus

had

an

advantage over the

challenge virus, either in time or in multiplicity of infection.

Adsorption,

penetration, and uncoating of challenge virus did not appear to be

in-hibited,

but the

synthesis of infectious viral ribonucleic acid

of the

challenge

virus

was

significantly retarded. It

was

shown with

temperature-sensitive viruses or mutants

that

the

replication

of viral ribonucleic acid by the interfering virus was required to

establish

interference. A mechanism of interference based on a competition for

replication sites or substrates is compared with other possible explanations.

The practical and theoretical

implications

of

viral

interference have stimulated

considerable

interest in this research

area.

Many reports have

been

published

since the

discovery

ofinterference in

plant

viruses

(14)

and in

animal viruses

(11).

Those papers that appeared

prior

tothe

discovery

of interferon

(13)

have been reviewed

extensively

by

Henle

(10)

and

Schlesinger

(18).

The role of

interferon

as an

important

factor inthe

develop-ment of

nonspecific

resistanceof the host cellsto

superinfection

with a second related or unrelated

virus is now

firmly

established

(6).

It is

presently

difficult

todeterminewhich of the

early reports

on

viral interferencewereconcerned with interferon

production

by

the hostor

by

otherfactors. It is

apparent,

however,

that there areseveral

types

of

viral interference

that do not involve

the

media-tion of interferon

(5, 7,

12, 15-17).

This report

describes anoninterferon-mediated

interference

among

different

arboviruses;

this

interference

requires

that the

interfering

virus be able to

replicate

viral ribonucleic acid

(RNA)

in

the host cell beforeit caninterfere withthe

growth

of the

challenge

virus.

Further,

interference of the

challenge

virus

appears

to occurafterits

uncoat-ing

but before

synthesis

of infectious viralRNA.

MATERIALS AND METHODS

Virus strains. In mostof the experiments

reported

here, the virus used to induce the interference was either the Trinidad strain of Venezuelan

equine

encephalitis (VEE) virus or strain T, which is a

small-plaque,

temperature-sensitive

variant of this virus. In a few

experiments,

the

interfering

viruses were: a large-plaque revertant ofT,

designated

V5,

whosemaximum

growth

temperaturewas

unchanged

(S. Halle, personal communication); an attenuated variant

(A)

of VEE virus

originally

described

by

Berge et al. (1); and atemperature-sensitive mutant (Ets-4) of the Louisiana strain of eastern equine

encephalitis virus (EEE). EEE virus served as the

challenge virus in most experiments. Properties of

these viruses, except for Ets-4 and V5, have been

describedby Brown (3).

Cellcultures. Cell cultures were prepared from

10-day-oldchickembryos. Thechick embryo (CE)

mono-layersweregrown,inlactalbuminhydrolysate medium

containing 10%calf serum, for24hr at 37 Cbefore infection. Details of the preparation ofmonolayers,

medium, and growth conditions were described in an earlier paper(22).

Virus growth. Except wherenoted, CE cell

mono-layersin 60-mmplasticpetri disheswereinfected with a virus seed preparedas a 10% CE seed, or as un-diluted tissue culture fluids obtained from infected

CEmonolayers.Theviruswasallowedtoadsorbfor 15 min at room temperature; thecells were washed twice withphosphate-buffered saline (PBS)atpH 7.4 to remove excess unattached virus, and were over-layeredwith5mloflactalbuminhydrolysatemedium

containing 10% calf serum. The cultures were

in-cubatedat37 Cina5%

C02-95%

airincubator.

When the

interfering

and challenge virus were

simultaneously added to CE monolayers, the virus

seedsweremixed beforeinfectionof the cells. When the challenge virus was added at some time after

infection of the cells with the

interfering

virus, the culture medium was removed, the challenge virus

wasadded, and,aftera 15-min adsorption periodat room temperature, the infected cells were washed twice withPBSandoverlayeredwith thelactalbumin hydrolysate medium. The cultures were reincubated at 37 C andsamplesof the culture mediumwere col-lectedatvarious intervals.In mostoftheexperiments, actinomycin D (1 or 2

,Ag/ml)

was incubated with cells for30min before infection andwasinthe culture medium at the same concentration

throughout

the

experiment.

Virusassay. Virusassayswereperformedon24-hr 1283

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CE monolayers prepared from 10-day-old chick embryos. The overlay medium was lactalbumin

hydrolysate-agar (21). In those studies involving mixed infection with VEE and EEEviruses, titers in the growthmedium were determined in thepresence

ofa 1:100dilution of anti-VEEserum (whose plaque

neutralization titer exceeded 1:10,000) added to the

agaroverlaymedium.PlaqueformationbyVEE virus

was inhibited but that of EEE virus was not. This permittedassay ofEEE virusgrowth in thepresence

ofa large excess of VEE virus. When strain T was

used, it was not necessary to add antiserum to the overlaybecause this virus formed very smallplaques and waseasily distinguishable from EEE virus when

assaysweremade onsamplesfrom mixed infections.

In reconstruction experiments involving mixtures

andcontrols,itwasfoundthat 100-to200-foldexcess

of T over EEE did not inhibit plaque formation of EEE, thusjustifying the procedure ofplaquing and countingEEEinthepresenceofexcessT.

Extractioni and assay ofinifectious RNA (IRNA). InfectedCEmonolayers wereremovedwitharubber

policemanand suspended in 0.02 M phosphate-0.001 M ethylenediaminetetraacetic acid buffer (pH 7.4).

Thecellswereextracted twice withcoldphenol (4 C), and theviralRNAwasprecipitatedfrom theaqueous

phase with three volumes of9570 ethyl alcohol

con-taining 2.0%// potassium acetate. The precipitate was

dissolvedin PBS, andthe IRNA wasassayed onCE monolayers treated with 1.0NI NaCl in a 0.1MI

tris-(hydroxymethyl)aminomethane-chloride buffer (pH 8.3) bythemethod of Colon and Idoine (4).

RESULTS

Demiionstration of interference. Interference of thechallengevirus could be demonstrated intwo

ways:(i) by infectingcells withVEEvirus several hours beforesuperinfectingthecultureswithEEE virusatmultiplicities equaltothose used forVEE

virus, and (ii) by infecting the CE cells

simul-taneously with two viruses at different

multi-plicities; the interfering virus was added at an

input multiplicity of about 10 plaque-forming

units (PFU) percell, whereas thechallengevirus

wasusedatamultiplicityof 0.1 PFUpercell. When equal multiplicities of the two viruses were employed, the degree of interference was

dependent upon the time of superinfection with thechallengevirus(Table 1).Thedegreeof inter-ference increased with the time that elapsed

be-fore superinfection with the second virus. Maxi-mal inhibition of the growth of EEE virus was observed when it was used to superinfect cells 5

to6 hr after infectionwith T virus.

The effect ofinfecting CEcells simultaneously with strainT and EEEvirus isshown inTable 2.

Strain T was addedata constant multiplicity of infection (MOI) of 10 andEEE viruswasadded

at an input MOI ranging from 1.0 to 0.01. The previous experiment and others showed that simultaneous infection at equal input

multiplici-TABLE 1. Effect of timne of superinfection on initer-ference with challenzge virius

Growth response ofchallenge Timeofsuperinifection virus(EEE)

with 11EEv-irusa

I'lFUml

Logioinhibition

hr-0 7.7 X 109 0

1 3.1 X 109 0.4

2 1.9 X 109 0.6

3 4.5 X 108 1.2

4 1.9 X 108 1.6

5 2.5 X 107 I 2.5

6 9.0 X 106 2.9

7 1.1 X 107i 2.9

aCultures were infected with strain T virus at

an input MOI of 10. Cultures were then washed two times with PBS and overlayered with culture medium. At the indicated times, themediumi was removed and the cultures were superinfected with EEE virus at the same multiplicity; the cultures were then washed twice and overlayered with

growth medium.

ICultures were held at 37Cfor 24 hr after

addi-tion of the challenge virus before virus assays were made.

cThis 24-hr titer was approximately the same

for all theEEE singly infected control cultures.

TABLE 2. Effect ofiunput muiiltiplicity ofthe

challenige

vi-rus

oln

the

in tee

fierence

wvit/i challen1ge

viruis

Inputmultiplicity PFU/m (2- hr)

Lo-io

irlhibition ofchallengexvirus'i E/i(4ucio

Controlb 1.9 X 10] 0

1.0 4.0 X 108 0.7

0.1 4.7 X 107 1.6

0.01 5.1 X 10- 2.6

Multiplicity of the interfering virus (strain T) was heldconstant at 10 PFU/cell.

bThe 24-hr control value was the growth re-sponse of EEE virus in the absence of strain T; this was approximately the same for each of the multiplicities tested.

ties of 10 resulted in no interference of either

virus;

multiplicities

higher

than 1 were therefore omitted.Theresults showeda

progressive

increase in the degree ofinterference as the multiplicity of

EEE virus decreased. Maximal

interference

was observedwhen thelowest

multiplicity

ofchallenge virus was used. The effect of

multiplicity

in this

experiment

wasprobablyonlyanother manifesta-tion oftheeffect of

time, since,

atthelower multi-plicities, more than one cycle ofchallenge virus

multiplication

would be required to reach the levels obtained by the interfering virus in one

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cycle. Inthe absence of strain T, EEE virusgrew

normally andto high titer; although the dataare notshown, therewas nointerference with T virus

growth in such doubly infected cells.

Therewas astronginhibitionof the growth of thechallenge viruswhen theinterfering viruswas

givenagrowthadvantage inthe CEcells either by

being inoculated several hours earlierorata

sig-nificantly higher MOI than the challenge virus (Tables 1 and 2). If the interfering virus was

treated with specific neutralizing antiserum just before infecting CE cells, interferenceto

superin-fectionwiththechallengeviruswasnotobserved. Controls consisting ofneutralizing antiserum to EEEvirus,ornormalserum,when incubated with

VEE as interfering virus prior to infection, did not preventtheinterference. These results showed

that infection bythe virusparticlewasnecessary

toestablish interference and that the interference

was probably not dueto interferon in the virus

suspension. The latter conclusion was supported

by the fact that virus that was sedimented and washed twice had the same interfering capacity

as crude virus.

Interference inducedbydifferentstrainsofVEE virus. Interference of the growth of EEE virus could also be demonstrated when other strains of VEE virus wereused. In addition, EEEvirus

could be used as the interferingvirus and could

inhibit the growth ofany strain of VEE virus.

However, there seemed to be some variation

amongvirus strains in theircapacity to serve as interfering viruses (Table 3). Strain A was

con-sistentlymoreeffectiveasaninterferingvirus than wasthe Trinidadstrainortheothers;theTrinidad strain was more effective than either T or V5;

therewasnosignificantdifference betweenTand

V5. The differences in the degree of inhibition induced by different strains may possiblybe ex-plained bydifferences in thecapacityof the

[image:3.484.251.444.82.216.2]

differ-ent virus genomes to attach to replication sites

TABLE 3. Capacity of differentt straintsofVEE virus tointerfere with thegrowth ofEEE

virus inchick embryo cell culture

VrEE virusstramna Degree of EEE virus_{interferenceb}

A 2.4

Trinidad 1.6

T 1.4

VS 1.1

aInput MOI, 100PFU/cell.

bInput MOIofEEEvirus, 1 PFU/cell.Thedegree

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of interferenceisexpressedaslog,0units ofEEEvirus at20 hr. Valuesareaveragesof fourexperiments.

TABLE 4.

Effect

ofactiniomycinz D oni

interferenice

with EEE virus multiplication by strain Ta

EEE virus (PFU/ml)

Time Sri

Strain T+ Stai T+ EEEvirus

EEE virusb EEEvirus alone

EE actinomycinD~' aln

hr

0 1.2 X 105 1.2 X 10' 1.2 X 105 6.5 4.1 X 106 6.4 X 106 6.0 X 107 22 4.7 X 107 4.6 X 107 2.4 X 109

aActinomycin D (1.0

gg/ml)

added at 2 hr

before infection.

bInfection by strain T followed by EEE 3 hr laterat equal multiplicities (100).

within

the hostcellorby

differences

in their rates or extentof

viral

RNA

replication.

Is the interference of virus growth mediated by interferon?

Actinomycin

D wasused to help

ob-tain evidence

onwhether

interferon

was a factor

in

the

interference

that was observed. This drug is

known

to

inhibit both

the

formation

and action

of

interferon in virus-infected

cells, yet it does not interfere with the synthesis of many RNA viruses (8, 20). If

interferon

were

involved

in the inter-ference observed here, then the challenge virus

should be able

to

multiply

normally in

the

pres-ence of

actinomycin

D. When

actinomycin

D

(1

Ag/ml)

was

added

2hr

prior

to

infection,

it had

noeffectonthe

interference

ofEEE

virus

in cells that had been

previously infected

with a high

multiplicity

of

strain

T

virus (Table

4). In both

the presence and absence of

actinomycin

D, the growth of the

challenge virus

was

inhibited

to the

same extent, about 1.6

log10

less

than that

ob-tained for

the

control culture.

Thatthe

actinomy-cin D was

active

was shown

by

the

fact

that growth of

vaccinia virus

was

inhibited

by more than99%

in

CE

cells

in

thesame

experiment.

In

addition,

the

drug abolished

the

interference

re-sulting

from

added

chick interferon (50

plaque-inhibiting units) in

a

Sindbis virus-CE cell

test system.Fromthese

results, it

seems

likely

that the

interference observed

does not result from the

formation

or

action

of

interferon

by the host cell. Subsequent

experiments

were

carried

out in

the presence of

actinomycin

D,andby infecting

with

equal

multiplicities

of the

viruses

3 to 4 hr

apart.

RNAsynthesis by the challengevirus.The inter-ference ofEEE virus growth that resulted when

strainT wasinoculatedontoCE cells4 hr

earlier

is shown in

Fig.

1.TheEEE

virus titer

wasreduced

2.7

log1o

below

that observed forthe control

cul-ture. In

doubly

infected

cells,

the synthesis of

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vitz and Brown, unpublished data). EEE virus, on the other hand, replicated normally at this

tem-perature. It waspossible, therefore, to inhibit the 7 ~~~~2-7Loglo growth of

strain

T

selectively

by

incubating

in-I fected CE

cultures

at 42 C

prior

toand after the

cultures were superinfected with EEE virus.

Figure

2

shows

the

effect

of

incubating doubly

J Z j K

infected cultures

at 42 C. In

this

experiment, T

/o/x,

viruswas

adsorbed

to

cells

atroom

temperature

2.7

Log10

washed

as

usual,

and

incubated

at 42C

for

3 hr

- ,,

before superinfecting with

EEE

virus

and

incubat-X

f'

fl t

ing

further at 42

C.

The interference normally

rx--X-x--- ' observed was largely abolished;i.e., the maximum /

.',,'

titer

of EEE

virus

was not

inhibited.

These results

suggestthat, as a

minimum condition

to

establish

interference,

the

interfering

virus RNA must be

able

to

replicate in

thehost

cell in order

to

inhibit

thegrowthof the challenge

virus effectively.

25 The

results

discussed

above

support

the notion

5 10 15 20 25 30 that a

competition

for

replication

sites or sub-Hours stratesaccounts for the interference observed. To 1.

Interference

of challenge virus (EEE) explore this idea further, a recently isolated tem-andinfectious RNA synthesis by strain T virus. perature-sensitive mutantof

EEE

virus was

em-infected at equal multiplicity (10 PFU/ml).

ployed.

At 37C, but not at 30 or 42

C,

Ets-4virus ige virus was addedto the culture3hrafter infec- exhibited an

unusually

high rate and extent of

ith strain T virus. Symbols:

(@-*)

singly viral RNA

synthesis;

itinduced the formation of IEEEvirustiter; (X-X)

doubly

infectedEEE approximately three times the amount of viral iter; (0-

-0)

singly infected EEE IRNA; RNAcompared to the parent, but produced

90%

()doublyinfected EEE IRNA. lessinfectious virus and at least

50%7

less

comple-ment-fixing

antigen

(Zebovitz

and

Brown,

unpub-IRNA

was

reduced

to

the

sameproportion as

that

of the mature

virus.

Theseresults suggest that the

interference

phenomenon

involves

a very

early

step

in

the

synthesis

of the

challenge

virus,

prob-ably

before

the

virus

has

had

the

opportunity

to

synthesize

its

IRNA. On thebasis ofourlimited

data

(Fig. 1),

there appears to

be

no

obvious

preferential interference between

IRNA

synthesis

and

viral

structural

protein

synthesis; otherwise,

the curves

of

virus interference and

IRNA

synthe-sis

would

not be

expected

to

parallel

each other. However,

since

no

direct

measureof

viral

protein

synthesis

was

made

independent

of

infectious

virus,

this conclusion is tentative.

Apparently,

the

early

step of the

challenge

virus

infection

that was inhibited was not adsorption,

penetration,

or

uncoating

of the

virus

genome, because interference was ofthe same

magnitude

whenIRNA of the

challenge virus

wasusedin the

place

of

infectious virus.

This

conclusion

receives

additional

support fromsome

incidental evidence

obtained

in an

experiment

(described

below) with

the T strain as

interfering virus

at 42C.

In

(22),

itwas

established

that the RNA of VEE

virus

enters thecell and

is

main-tained

in a

viable

state, even for

prolonged

periods

at 44C,

although

nonewRNA

is

synthesized.

The same

proved

true forstrainT

virus

at42C

(Zebo-10 .

9 10

E

"I

c c

r-8 10

7 10

6

10

105[

104

3

10 2

0 ~~~~~~~0

.1,

11

00s

X"

0 5 10 15 20 25 30

Hours

FIG. 2. Effect of incubationat42 Con thecapacity

of strain T virus to interfere with EEEvirusgrowth.

Challenge virus (EEE) wasadded3hrafter infection of CE cells with strain T. Symbols: (0) EEE virus

growthinabsence ofstrain T; (X) EEE virus growth

onCEcells infected with strain T. (0) StrainT virus

growth inabsenceof EEE virus. 10

10 9 10

8-10

7-7

- 10

°

106

10

.t

5

.2 10

10 2

3:10

10

FIG. growth Viruses

Challen

tion wi infected virus ti (X-->

Yr%'ITA

1286

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TABLE5. Comparison of Ets-4andEEE as interfering virus

Time aftersuper- Control VEE EEE+VEE Logioinhibition Ets-4 + VEE Logioinhibition

infection' (PFU/ml) (PFU/ml) (PFU/ml)

hr

0 7.1 X 104 6.0 X 104 5.1 X 104

5 1.5 X 106 4.0 X 105 0.6 1.0 X 105 1.1

8 2.0 X 107 3.0 X 106 0.8 1.9 X 105 2.0

10 2.0 X 109 7.2 X 107 1.6 3.5 X 105 3.8

a Cellswereinfectedwith eachvirusat anMOIof 10, 3 hr apart.

lisheddata). Because thismutantproduces larger

amountsof viral RNA than does theparent, one

might predictthatmorereplicationsiteswouldbe

occupied and therefore agreater degreeof

inter-ference should result when it, instead of the parent, is usedastheinterferingvirus. Ets-4was indeed a much better interfering virus than its parentwhenVEE was used as a challenge virus

(Table 5). From doublyinfected cells,VEEvirus

wascounted in thepresenceofa 1:100 dilution of anti-VEEserum(titer,1:100,000).Theinterfering

virusinthis experimentwasincubatedat37 C for 3 hr before challenge virus was added. If the

initial 3-hr incubation was carried out at 30 or 42 C,at whichtherate andextent ofviral RNA

synthesisof Ets-4were depressed to levels closer to those found in the parentvirus,the degreeof

interference was likewise reduced (Table 6). It was notreducedto the level inducedbyEEE

be-cause, when incubation was resumed at 37C afteronly3hrateither of the other temperatures,

Ets-4 stillhadanadvantagein theextentofviral RNAsynthesis.

DISCUSSION

Several types of interference among viruses have been described (2) that do not involve the

mediationof interferon. Except forafew reports

(16, 19), all of the interference phenomena de-scribedrequiredthat theinterferingvirus be able to initiate certain synthetic processes in thehost cell. To demonstrate the interference between

arboviruses, itwas necessaryto givethe

interfer-ing virus a growth advantage either by previous

infectionof the host cells orby using high multi-plicities of the interfering virus relative to the

challengevirus. Thegreater thegrowth advantage given to theinterfering virus, the greater the

de-gree of interference. The interference, however,

didnotresult fromageneraldeterioration ofcell

metabolism due to infection by the interfering

virus. At least 40 hr ofinfection (at multiplicities

greater than 1) with EEE or VEE viruses was

requiredfor the cellsto showa cytopathic effect, even though peak titer was attained between 10 and 12 hr. Furthermore, in our hands these

TABLE 6. Effect of initialtemperature of

intcubation

on interference

intduced

by Ets-4virusa

Initialtempofincubationb

Time after superinfection

37C 30C 42C

5 1.4 0.9 1.0

8 2.2 1.2 1.4

20 3.8 2.8 2.9

aExpressed as the

log,0

decrease of VEE virus

in doubly infected cells compared to controls incubated with VEE virus alone.

bCultures infected with Ets4 virus were incu-bated at 37, 30, or 42 C for 3 hr prior to

super-infection with VEE virus. Incubation was then resumed at 37 C for all cultures.

viruses

cause

little

or no

inhibition

oftotal RNA

or

protein synthesis

in

monolayer

cultures of CE cells forat

least

12 hr

after

infection.

In contrast to the

interference described

by

Pohjanpelto and

Cooper

(16),

the presence of the

virus

genome

in

the

cell

without

accompanying IRNA

replication

inoursystem was not

sufficient

to

induce interference of

challenge

virus growth. When the

growth

of

strain

T was prevented by

incubation

at 42 C, the growth of the challenge

virus (EEE)

was not

inhibited.

It

is known

that the

block in

the

growth of

T

virus

occurs

because

synthesis

of IRNA

is inhibited

at 42 C. These data therefore

indicate

that the

interfering virus

genome must

be able

to

replicate

IRNA

in

the cell

in

order to prevent the

growth

of the

chal-lenge

virus.

Thedata

above

appeartosupportthe

hypothe-sis

of Cords

and Holland

(5)

that

interference of

challenge virus

occursbecause of

competition

for replication sites or substrate necessary for viral replication. This

hypothesis

wasfurther strength-ened

by

showing

that Ets-4was abetter

interfering

virus

than was EEE at 37C, a temperature at

which it

produces

threetimesasmuchviral RNA

as does theparent EEE

virus.

At

initial

tempera-turesof

incubation

of 30 and42C,at

which

Ets-4

virus

begins

to

produce

viral RNA at rates

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ZEBOVITZ AND BROWN

proaching

that of EEEvirus, Ets-4 interferes toa lesserextentwith VEE virus growth thanat37 C.

Interference of challenge viruswasobservedin

one experiment in whichsuperinfection was car-riedoutwith the IRNA of challenge virus instead of thevirion, and inasecondexperiment in which

the inhibition of IRNA synthesis of challenge virusaftersuperinfection with the virion was evi-dent. Itseems reasonable toconclude, therefore, that interferenceprobably occurredatsomepoint after theentryanduncoatingof theviralnucleic

acid but beforetheinitiation of IRNAsynthesis. This conclusion is similar to that reported by Cords andHolland (5) forenteroviruses.

TheevidenceontherequirementforviralRNA

synthesistoestablishinterference, and the

inhibi-tionofsynthesisofIRNA ofchallenge virus,

sup-portthe interference mechanismbasedon a

com-petition for replication sitesormetabolites. Inter-ference of this type, although less directly stated

or supported, has beendescribed forarboviruses by Henderson and Taylor (9) andbyP.T. Allen

(unpublished data). Recent preliminary

experi-mentsindicate that various arbovirusescan

inter-fere with Newcastle disease virus and vesicular stomatitus virus, and vice versa, in the presence ofactinomycin D. We have not,however,

elimi-nated adsorption, penetration, or uncoating of

challenge virus in these combinations, nor have weyetfollowed viralRNA ofchallenge virus in

these systems. If, infact, the actinomycin

D-re-sistant interference isasbroadassuggested above, the major hypotheses reviewed by Bratt and Rubin (2) as alternatives to the competition hy-potheses would be eliminated because of the rela-tivespecificity required. Further experiments are needed, not only to determine how broad is the

interference

described here, but also to provide direct proof where possible of the competition hypotheses.

If the kind of interference described in the

present paper is found to extend nonspecifically

tounrelatedviruses, then its role in vivoas a

non-specific mechanism ofresistance to virus disease would have to beevaluated, particularly in rela-tion to interferon-mediated interference. The

latter isusuallyassumed toplayadominant role

inmostofthein vivo interference that hasbeen described before and after thediscovery of inter-feron.

ACKNOWLEDGMENTS

ThispaperisdedicatedtoStewartA. Koser,onthe

occasion of his 75th birthday, in recognition ofhis distinguishedcareerinteaching andresearch.

Weexpressappreciation fortheexcellent technical assistance of Virginia Elsner, Delbert Davisson, Rosa Bell, and Cordia Harris.

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