UNCLASSIFIED

AD NUMBER

AD844170

NEW LIMITATION CHANGE

TOApproved for public release, distributionunlimited

FROMDistribution authorized to U.S. Gov't.agencies and their contractors; CriticalTechnology; OCT 1968. Other requests shallbe referred to Commanding Officer,Department of Army, Fort Detrick, Attn:Technical Releases Branch/TID, FortDetrick MD 21701.

AUTHORITY

Army Biological Defense Research Lab ltrdtd 29 Sep 1971

THIS PAGE IS UNCLASSIFIED

AD *0

TECHNICAL MANUSCRIPT 477

INTERFERENCE AMONG

1- GROUP A ARBOVIRUSES6>0 .&...3

Eugee Z0ovit

Arthur Brow

009itA 0

vi: i.A

A00 B0

DEPATMEN OFTEAM

For Detrick

ThisI 2oueti'sbett peileprcotrl an ec tasmttltofreg

govenmets r faignnatonal ma bemadonly wihpio proa f et o ry

For Derik ATTN: OTOeRhia Reeae68ach*I. Frdrik Marlad 270

Reproduction of this publication in whole or inpart is prohibited except with permission of theCom-.nding Officer, Fort DeLrick, ATTN: Technic•Sa"Releases Branch, Technical Infori-acion Division,Fort Detrick, Frederick, Maryland, 21701. However,DDC is authorized to reproduce the publication forUnited States Government purposeE.

DDC AVAIlABIUTY NOTLCES

Qualified requesters may obtain copies of thispublication from DDC.

Foreign announcement and dissemination of thispublication by DDC is not authorizeL.

Release or announcement to the public is notauthorized.

DISPOSITION INSTRUCTIONS

Destroy this publication when it is no longerneeded, Do not return it to the originator.

The findings in this publication are not to beconstrued as an official Department of the Armyposition, unless so de'ignated by other authorizeddocuments.

I =

A_ k 2 -- t. .

r H

DEPARTMENT OF THE ARMYFort Detrick

Frederick, Maryland 21701

T•CHNICAL MANUSCRIPT 477

INTERFERENCE AMONG GROUP A ARBOVIRUSES

A

Eugene Zebovitz

Arthur Brown

• Virus and Rickettsia DivisionS~BIOLOGICAL SCIENCES LABORATORIES

Project l8014501B71A October 1968

IiA-

A - - -• •; o : _ . T• ---..- - -, - -• ' - ---- :----- -"

I "

11 2

SInterference among group A arboviruses that did not involve

__• .ithe mediation of interferon is described, Interference wasobserved only if the interfering virus had an advantage overthe challienze virus either in time or in multiplicity of

infection. Adsorption, penetration, and uncoatin8 of challengevirus did not appear to be inhibited J but the Byntheels ofinfectious viral RNA of the challenge virus was significantly "retarded. With temperature-sensitive virures or mutants•the replication of viral RNA by the interfering vi-rus was -

I • ~ required t~o establish interference. A mechanism of inter-

ference based on a competition for replication sites orsubstrates was compared with other possible explanation~s.i

I1

2~ IB-F e

II

3

CONTENTS

Abstract . ..a. .. .a...... . . . . 2

I. INTIODUCTIOlN . . ......... . a . a. ... . 5

II, MATERIALS AND METHODS .a. . . . . . . . . . . . . . . . . . . . . 5A, Virus Strains .. a . .. . . . . . . . . 5B. Cell Cultures . .a . . . . . .a a a a a. a a. .. . . 6C. Virus Growth a A... aa...a......aa..... 6D. Virus Assay . . . . . .a . . . . . . . . . . . . . 6E. Extraction and Assay of Infectious Ribonucleic Acid .. a 7

IIl. RESULTS . . . . . . . . . . . . . . ..t 7A. Demonstration of Interference . .a.a.. ... .. . 7B. Interference Induced by Different Strains of VER Virus ... a 9

Co Is the Interference of Virus Growth Mediated by Interieron . 10D. IRM Synthesisb y the Challenge Virus .. .a . . . .... . 11

IV. DISCUSSION . a a a a . a . . a . a a a a a a a a a a a a a a a . a 16

Literature Cited . a a a a a 19Distribution List a.a.a. a .21Form 1473 .. a a .. .. . , .. . . . . . . 23

FIGURES

1. Interference vMth Challenge Virus (EM) Growth and 1MSynthesis by Strain T Virus . . a........a... .. a .12

2. Effect of Incubation at 42 C Upon the Capacity of Strain TVirus to Interfere with EEN Virus Growth ........ ............. 14

TABLES

1. Effect of Time of Superinfection on Znterference with ChallengeVirus .a a a .a a 8

2. Effect of Multiplicity of the Challenge Virus on the Interferencewith Challenge Virus . a a a .a .a a P 9

3. Capacity of Difftrent Strains of VIE Virus to Interfere with theGrowth of UE Virus in Chick Embryo Cell Culture. . ...... . 10

4. Effect of Actinowycin D Upon Interference with ZEE VirusMultiplication by Strain T . .. .. ... . .. a.. . 11

;JN

5. -Comparison qf. Zts-4 and MEEax Interfering Virus ..... . . 156. Effect of Imitial Temperature of Incubation on Interfer~ence Induced

by Etv-4 Virus . . . . . . . . . . . .15

4ý

4-

I. INTRODUCTION

The practical and thearetical Implications of viral interf ezence havestimulated considerable interest in this research area. Many reportshave been published since the discovery of interference in plant viruses2

and in animal viruses A Those papers that appeared prior to the -discovery of interferorP have been reviewed extexsively by lienle4 and bySchlesinger.-6 The role of interferon as an Important factor in thedevelopment of nonspecific resistance of the host cells to superinfectionwith a second related or unrelated virus is now firmly e~stablished~.6

It is presently difficult to determine which of the early reports onviral interference -were the result of interferon production~ by thehost or of other factors.. It is apprent,, however, that there areseveral types of viral interference that do snot involve the mediationof inefern7

The present report describes an interference not mediated byinterferon among different arboviruses that requires that the inter-fering virus -replicate viral AlNA in the host cell before it -canfnerfere withthgrwho the challenge virus. aprstocuaFuther, itunctetr-ineferene withtegrtho the challenge virus aprstocuafutherIt incoter

but before synthesis of Infectious vital SM&.K

II. MATMRALS AND METHODS

A. VIRUS STRAINS

In most of the experiments-reported here, the virus used to Inducethe Interference was either the Trinidad strain of Venezuelan equineencephalitis (VEN) 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 of T, desig-nated V5, whose maximum growth temperature was unchaniged;* anattenuated variant (A) of VEE virus originally described by Berge,Banks, and Tigertt;213 and a temperature-sensitive mutant (Ets-4)of the Louisiana strain of eastern equine encephalitis virus (REE).2-*-**EEE virus served as the challenge virus in most experiments. Propertiesof these viruses except for Ets-4 and V5 have been described by Brown? 52rThe special properties of Its-4 pertinent to its use are described inSection III of this report.

*Halle, Si., personal coiununicstion.A~Unpublished data.

ok~ - _ER

'1 6

I. CEL CUTURECell cultures vere prepared from 10-day-old chick embryos. The chick

embryo (CE) mnonolayers were grown in lactalbumin hydrolyzate mediumcontaining 1.0%,calf serwu for 24 hours at 37 C before infection. 'Detailsof the preparation of monolayers, medium, and growth conditionsv~erej -described in an earlier paper 286

ZIVRSGOTZxcept wmhere nioted, CE cell monola-yera in 60-mm plastic petri dishes

-were -infected -with a -virus seed prepared as a 10%. CE seed or undilutedtisisue culture f luids obtained from infected CE monolayers. The -virus-was allowed-to adsorb for 15 minutes at room temperature; the cells

ieere then -washed -twice with ?hosphate buffered saline (PBS) at pH 7.4

hydr-olyzate medium. with 10% calf serum. The cultures were incubated at-j 37 VC -u a 5% CO2 - 93% air incubator.

I-n those experiments where the Interfering and challenge -viruses -wereadded simultaneously to CE -monolayers, the -virus seeds were mixed bef oreinfection-of the cells. When the challenge virus was added at some time

j ~after infection -of the cells with the interfering-virus, the culture~medium itas removed,, the challenge viu wsadd nafter a 15-minuteads orption period at room temperature, the i-nf ected -cells were -washed

,twice with MiSand overlayed with the lactalbumin bydrolyzate -medium.¶The -cultures -were ireincubuted at 37,C and samples ýof the culture medium

- were collected at various Intervals. lInmost of the experiments, asIndicated In Section 11I, actinom~ycin D at 1 or 2 iig/Ml -was, preincubatedvith -cells for .0.5 hour- b --f ore inf ection and vwas held at the sameýconcentration tbroug'hout the experiment.

D. D VIELUS ASSAY

NVlx; assays were performed on 24-hour CE monolayers prepared from --

10-&- y-old chick embryos. The overlay medium was lacta-lbumint hrolyzate

-with VEXand ZEE viruses,, titers in t-he supernatant growth -medium -were

determined In the presence of a 1.-100 dilution of anti-VEX serum (whosepl1aque n~eutralization titer exceeded 1:10, 000) added to the agar overlaymedium. Plaqjue formation by VTEE virus -was Inhibited but that of LEE-virus was not. This permitted assay ýof IEEE virus growth in the presence-of a large excess of VIFE -virus. 'When strain T was used, it -was notnecessary to add -ar iserum to the -overlay because this -virus formed verysaill plaques and 'I as easily distinguishable from ZEE virus -when assaysVere nade on samp. ts from mixed infections. In reconstruction experiments

AA-~_ ___Z _

WIN

7

involvtng mixtures and controls, 100- to 200-f old excesses of T over[RU~ did -not Inhibit plaque formation of BEEE, thus justifying the

procedure of plaquing and counting iEEE in the presence of excess T.

E. BXTRACTION AND ASSAY OF INFECTIOUS RIBONUCLEIC ACID

Infecied CE monolayers were removed with a rubber policeman andwere surpanded in 0.02 14 phosphate - 0.001 N ethylenediaminatetraaceticacid -META) Ibuffex, pH 7.4. These suspensions were extracted twice-with -cold (4 C) -phenol, and the viral RNA was precipitated :from theaqueoua phase with -three volumes of 95% ethyl alcohol containing 2.0%potassium -acetate.. The precipitate was dissolved In Z~BS, and the inf ec-tious ribonucleic acid (IMN) was assayed on CE monolayers treatedwith 1.0 -1 NaCI in a 0.1 Xl Tris-'HCi buffer at pH 8.3 according to themethod described by Colon and Idoine.2

8

III-. RESULTS

A. DEMONSTRATION OF INUERERECE.

Interference -with the challenge -irur~s ~could be demonstrated :intwo ay:(i) Iry infeacting cells -wit~h VI= vizrus several -hours 'bef oresuperinfecting -the -cultures -with ZEE virus at multiplicities -equalto those -used f or VTEE -vxr,, and (Ii) hy infeacting the CE cellssimultaneously with two vixuses at -different -multiplicities; theinterfer-ing vixus was added at an input multiplicity of about 10plaque-forming units per cell, while the challenge virus was used-at

a 100-fold lower multiplicity.IIn the case where equal multiplicities of the two viruses -were

employed, the degree of the interference was, dependent upon the timeof superinfection with the challenge -virus (Table 1).. The diegree ofInterference increased -with the time that elapsed before suiperinfectionwith the second virus. Maximum inhibition of the growth of iEEE virusvas observed -when it -was used to superinfect cells 5 to 6 hours '

&after infection -with 7 virus.. W

F&

8

TABLE 1. EFFECT OF TIME OF SUPERINFECTION ONINTERFERENCE WITH CHiALLElPCE VIRUS

TI~meGrowth Response ofTie fChallenge Virus E)ISuperinfectiona-/ with 24-Hour Virus Log,,

4 E Vrshur Titers, pfum/ Inhibition

I0 (Control)&S/ 7.7 x 109 011 3.1. x 10~ 0.4

2 1.9 X lo0 0.63 4.5 x108 1.24 1.9 x 108 1.6

,* I5 2.5 x 107 2.56 9.0 xc 10 2.9

7 1.1 x 107 2.9

Ia. Cultures were infected with strain T virus at an input "DOI ofj 10. Cultures-were then washed two times v~th PBS and overlayed

with culture medium. At the indicated time the medium wasIi removed and superinfected with ERE virus at the same

multiplicity; the cultures were then washed -twice and over layedwith. growth medium.

b, Cultures were held at 37 C for 24 hours after addition of thech~allenge virus before virus assays were zaade. [

c. This 24-hour titer was approximwately the same for all the BEEsingly infected control culturee.

T-he tsff~ect of infecting CE cells simultaneously with strain T andESE 71-ru~s 19 shotan in Table 2. Strain T was added at a constant

wa~ipicity of Infection (POI) of 10 and EEE virus was added at-input M07 ranging iroum 1.0 to 0.01. The degree of interference increasedpzograsa4tvely az the -mwitiplicity ef HEE virus decreased. Maximuminterference wai. observed when Tt 1c-est multiplicity of challengevirus was used. Xn the absenca of atxin T, BEE virus grew normallyand- to bigh titer,, and,~ho~ rhe date are not shown, there was no --

ifiterfei.'ence with T vir-aa grw-th in such dot'biy infected cells.

--At

e- -77Z'

-91

TABLE 2. EFFECT OF MULTIPLICITY OF THE CHALLENGEVIRUS ON THE INTERFERENCE WITH CHALLENGE VIRUS

Multiplicity of TiterChallenge Virus (EEE)!/ (24-Hour) Logl0 Inhibition

Contro1li' 1.9 X 109 0

1.0 4.0 x 108 0.7

0.1 4.7 x 107 1.6

0.01 5.1 x 105 2.6

a, Multiplicity of the Interfering virus (strain T) was heldconstant at 10 pfu/cell.

b. The 24-hour control value was the growth response of BEEvirus in the absence of strain T; this was approximately

the same for each of the multiplicities tested.

The data in Tables I and 2 show that there was a strong inhibition Iof the growth of the challenge virus when the interfering virus was given ta growth advantage in the CE cells either by being inoculated severalhours earlier or at a significantly higher MOI than that of the Ichallenge virus. If the interfering virus was treated with specificneutralizirg antiserum just before Infecting CE cells, interferenceto superinfection with the challenge virus was not observed. Controlsconsisting of neutralizing antiserum to BEE virus, or normal sertum,when incubated with VEE as interfering virus prior to infection, didnot prevent the interference. These results showed that infectionby the virus particle was necessary to establish interference and thatthe interference was probably not due to interferon in the virussuspension. The latter conclusion was supported by the fact thaat T'virus that was sedimented and washed twice had the same interferingcapacity as crude virus.

B. INTERFERENCE INDUCED BY DIFFERENT STRAINS OF VEE VIRUS

Interference with the growth of BEE virus could also be demonstratedwhen other strains of VEE virus were used. In addition, BEE viruscould be used as the interfering virus ar-d could inhibit the growthof any strain of VEE virus. However, there seemed to be some variationamong virus strains in their capacity to serve as interfering viruses.Table 3 shows the average results of four experiments. The degreeof inhibition of kEE virus induced by each virus strain varied. Among

-v - -. -~4

IM -- t~

10

ViE viruses., strain A was the most effective interfering virus,, followedby the Trinidad strain and finally strains T and V5. The differencesin the degree of inhibition induced by different strains may possiblybe explained by differences in the capacity of the virus genomes to attachŽ1to replication sites within the host cell and/or to differences in theirrates or extent of viral RNM replication (gee later discussion concerningEts-4 virus).

TABLE 3.* CAPACITY OF DIFFERENT STRAINS OF VIE VIRUSTO INTERFERE WITH THE G8014'X OF ESE VIRUS

IN CHICK EMMYO CELL CULTURE

VIEVirs srai~e/Degree of Interference in Loglo UnitsVEE irusStraof SEEl Virus Titer at 20 HourskI

A ~2 .40S

Trinidad 1.6

T 1.4

ai. VIE virus iftiinx at input -MOI of 100 pfu/cell.b-ENE vliru used. -at input MDI of I pfu/cell.

.c. Valueis are averages of four experiments.

Ci STHE INEFRNEOF VIRUS 'GROWT MEIATED BY ±INTMEROUg

P Actinownycin Dwas-used to help obtain evidence on whether interferonwasA f~id intheinterference that was observed. This drug is known

,to inhibit both the formation and action of interferon in virus-infectedcellsyti does not -interfere with the synthesis of many RNA virufles~

- f.,interferoh were involved in the interference observed here., the chailenge k

~ ~i~usshould be able to multiply normally in the presence of actinomycin D).Thbl~& -44w that actinomycin D (1 Im) when added 2 hours prior- ---

,tojinfection, had no effect upon the- interference with SEE virus in cellsthat had been previously infected with a highi multiplicity of strain Tvirus. In both the~presence and absence of actinomycin D), the growthof the challenge virus -was inhibited to the same exteniý, about 1. 6 logjaless than that obtained tor-the control culture. That the actinomycin DI -was active wits shown by 'the inhibition of growth of vaccinia virusby more than 99% in CE cells in the same experimant. In addition, thedrug abofished the interference resulting from added chick interferon(5-0 plaque-inhibiting units) in a Sindbis virus -CE cell test system.

- --

K,,-4

...-.-.-.-.-.-.-.-.-.-.

I

•J 11

From these results it seems likely that the interference observed doesnot result from the formation or action of interferon by the host cell.Subsequent experimente were carried out in the presence of actinomycin " -D and by infecting with equal muiltiplicities of the viruses 3 to 4 hoursapart.f

TABLE 4. EFFECT OF ACTINOMYCIN D UPON INTERFERENCEWITH ZEE VIRUS MULTIPLICATION BY STRAIN TYJ

S~Plaque-Forming Units/ml of gEE VirusTime, Strain T + Strain T + HEE EEE Virus

hours BEE Virus Virus + Actinomycin D•! Alone

0 1.2 x I05b/ 1.2 x I05Yk/ 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 10

a. Actinomycin D added at 1.0 gg/ml 2 hours before infection.B. Infection by strain T followed by gEE 3 hours later at equal

multiplicities (100).

I7>

D. IRNA SYNTHESIS BY THE CHALLENGE VIRUS

The interference with EEE virus growth that resulted when strain T ->was inoculated onto CE cells 4 hours earlier is shown in Figure 1. TheREE virus titer was reduced 2.7 log, below that observed for the controlculture. Superimposed on this curve are two curves for the synthesisof IM of the challenge virus. In doubly infected cells the synthesisof IRNA was reduced to the same proportion as that of the mature virus. "These results suggest that the interference phenomenon involves a veryearly step in the synthesis of the challenge virus, probably beforethe virus has the opportunity to synthesize its IREA. There appears

i to be no obvious preferential interference between LRNA synthesis andviral structural protein synthesis; otherwise, the degrees of the two in-hibitions would be expected to vary more significantly than was found.

t>

12

* 10

210 1

1 10

1 10 2.7 Logp

a 106

"0 0 10

44 4-210

IL,

11

00 5 10 15 20 25 30

Hours

Figure 1. Interference with Challenge Virus (ZEE) Growth and IRN&Synthesis by Strain T Virus. Viruses infected at equal M01(10 pfu/ml). Ch~allenge virus vas added to culture 3 hoursafter Infection with Strain T virus. Symbols: 0-@, singlyinfected EEE virus titer; X-X, doubly infected HEE virustiter; 0-4, singly infected EEE IRNA; X--X, doubly Infected

NE M

ýA.

13

Apparently the early step of the challenge virus infection that wasinhibited was not adsorption, penetration, or uncoating of the virusgenome, because interference was of the same magnitude when IRNA ofchallenge virus was used in the place of infectious virus. Thisconclusion receives additional support froa some inc'.dental evidenceobtained in an experiment described below using the T strain asinterfering virus at 42 C.

In previous studies,18 the RNA of VEE virus entered the cell andwas maintained in a viable state even for prolonged periods at 44 C,although no new RNA was synthesized. The same proved true for T virusat 42 C.* EEE virus, on the other hand, replicated normally at thistemperature. It was possible, therefore, to inhibit the growth ofstrain T selectively by incubating infected CE cultures at 42 C priorto and after the cultures were superinfected with EEZ virus.

Figure 2 shows the effect of incubating doubly infected culturesat 42 C. In this experiment, T virus was adsorbed to cells at roomtemperature, washed as usual, and incubated at 42 C for 3 hours beforesuperinfecting with FEE virus and incubating further at 42 C. Thus,the interference normally observed was largely abolished; i.e., themaximum titer of EEE virus was not inhibited. These results suggestthat, as a minimum condition to establish interference, the interferingvirus RNA must be able to replicate in the -host -41! in order to inhibitthe growth of the challenge virus effectively.

The results discussed above support the notion that a competitionfor replication sites accounts for the interference observed. Toexplore this idea further, a recently isolated temperature-sensitivemutant of EZE virus was employed. At 37 C, but not at 30 C or 42 C,Ets-4 exhibited an unusually high rate and extent of viral RNA synthesis:it induced the formation of approximately three times the amount ofviral RNA compared with the parent, but pr6duced 90% less infectiousvirus and at least 507% less complement-fixing antigen. 14,** Becausethis mutant produces larger amounts of viral RN& than the parent.,one might predict that more replication sites would be occupied andtherefore a greater degree of interference should result when it,instead of the parent, is used as the interfering virus. The resultspresented in Table 5 show that Ets-4 is indeed a much better interferingvirus than its parent when VEE is used as a challenge virus. Fromdoubly infected cells, VEH virus was counted in the presence of a 1:100dilution of anti-EEE serum (titer was 1:100,000). The interferingvirus In this experiment was incubated at 37 C for 3 hours beforechallenge virus was added. If the initial 3-hour incubation wascarried out at 30 C or 42 C, where the rate and extent of viral RNAsynthesis of Ets-4 was depressed to levels closer to those found in theparent virus, the degree of interference was likewise reduced (Table 6).

Zebovitz, E.; Brown, A. Unpublished data.** Unpublished data.

7;

S- -.

1" 14

10-10

9-10

S#

E 6

101C 10 ,-1 0= 10

o /a 2

0 10 --

••0 5 10 is 20 25 30 •

Figure 2. Eff~ect of Incubation at 42 C Upon the Capacity of Strain TVirus t~o Interfere with EEZ Virus Growth. Challenge vir~us .:(EEE) added 3 hours after infection of CE cells with Strain T.Symbols: 0-O, EEE virus growth in absence of strain T;.,.X--X, UFF, virus growth on CE cells infected w~ith strain T.6-,-t strain T virus gr~wth in absence of EEE virus.

Z-I

i•'"I HFiue2 Efc f nuaio t4 Uo hCct o tan

- 7- --

-~ -15

It wa no reue totelvlidce1yEZbcue. hnicbto

was esued a 37C ater nly3 hurs t ethe of ~e the

It was nfetot a reu ed , to he evl induced bynEhibecauon wenm Incubition

0a rseda37. C afte o 6ly 3 hour at1 eihe of14ohe

tepeaurs 5t- tl a navnaei h xeto ia N

0 ~ ..x 107 6.0 x 106110

510 2.0 x 109 7.2 z 107 1.6 3.5 x 10~ 3.8

a. Cells were infected with each virus at an MOI of 10, 3 hours apart.I

TABLE 6.* EFFCT OF INITAL TEMPERATURE OF INCUBATIONON INTERFERENCE INDUCED BY Sts-4 VIRUS

Hours DMEee of Interference with YEEA..After Initial Temperature of Incubation D

Superinfection 37 C 30 C 42C

5 1.4 0.91.

8 2.2 1.2 1.4~20 3.8 2.8 2.9

a. Logy, decrease of VRE virus titer in doubly infected cellsomipared with controls incubated with VEE virus alone. -

b. Cultures infected with Ets-4 virus were incubated at either37 C, 30 C,. or 42 C f or 3 hours prior to superinfectionAwith VEE virus. Incubation was then resumed at 37 C forall cultures.

--- :K11:ii

* 4~

{- .D. . . ......... . . ... .

1 16

IV. DISCUSSION

Several types of interference among viruses have been described (reviewedby Bratt and Rubin3 1) that do not involve the mediation of interferon.Except for a fe-w reports (eog., Pohjanpelto and Cooper, " Steck and RubinP2 )all of the interference phenomena described required that the interferingvirus be able to initiate certain synthetic processes in the host cell.To demonstrate the interference between arboviruses. it was necessary togive the interfering virus a growth advantage either by previous infectionof the host cells or by using high multiplicities of the interferingvirus relative to those of the challenge virus. The greater the growthadvantage given to the interfering virus, the greater was the degree ofinterference. The interference, however, did not result from a generaldeterioration ot cell metabolism due to Infection by the interferingvirus. At least 40 hours of infection (at multiplicities greater than 1)with LEE or VEe viruses were required for the cells to show a cytopathiceffect, even though peak titer was attained between 10 and 12 hours.Furthermore, in our laboratory these viruses cause little or no inhibitionof host-cell ENA or protein synthesis in monolayer cultures of CE cellsfor at least 12 hours after infection.

In contrast to the interference described by Pohjanpelto and Cooper, 1'the presence of the virus genome in the cell without accompanying IRNAreplication was not sufficient to induce interference of challenge virusgrowth in our system. When the growth of strain T was prevented byincubation at 42 C, the growth of the challenge virus (LEE) was notinhibited. It is known that the block in the growth of T virus occursbecause synthesis of LEN is inhibited at 42 C. These data thereforeindicate that the interfering virus genome must be ab1le to replicateIM in the cell in order to prevent the growth of the challenge virus.

The data above appear to support the hypothesis of Cords and Hollandlothat interference of challenge virus occurs because of competition forreplication sites or substrate necessary for viral replication. Thishypothesis was further strengthened by showing that Ets-4 vas a betterinterfering virus than EEE at 37 C, a temperature at which it producesthree times as much viral ENA as the parent LEE virus. At initialtemperatures of incubation of 30 C and 42 C, where Ets-4 virus beginsto produce viral RNA at rates approaching that of EEE virus,* Ets-4"- rferes to a lesser extent with VEE virus than at 37 C.

* Unpublished data.

-M-

17

Interference of challenge virus was observed in one experiment wheresuperinfection was carried out with the IM of challenge virus insteadof the virion, and in a second experiment where the inhibition of IU'A

synthesis of challenge virus after superinfection with the virion wasevident. It seems reasonable to conclude, therefore, that interferenceprobably occurred at some point after the entry and uncoating of the iiviral nucleic acid but before the initiation of IRNA synthesis. Thisconclusion is similar to that reported by Cords and Holland'0 forenteroviruses.

The evidence on the requirement for viral RNA synthesis to establishinterference on one hand, and the inhibition of synthesis of IENA ofchallenge virus on the other, supports the explanation that the interferencemechanism is based on a competition for replication~sites or metabolites.Interference of this type, although less directly stated or supported,has been described for arboviruses by Henderson and Taylor2 3 and byAllen.2' Recent preliminary experiments indicate that various arbovirusesinterfere with Newcastle disease virus and vesicular stomatitis virus,and vice versa, in the presence of actinomycin D. We have not, however,eliminated adsorption, penetration, or uncoating of challenge virusin these combinations, nor have we yet followed viral ENA of challengevirus in these systems. If, in fact, the actinomycin D - resistantinterference is broad as suggested above, the major hypotheses reviewedby Bratt and Rubin"' as alternatives to the competition hypotheseswould be eliminated because of the relative specificity required. Further

experiments are needed, not only to determine how broad is theinterference described here, but also to provide direct proof wherepossible of the competition hypotheses.

If the kind of interference described in the present paper is foundto extend nonspecifically to unrelated viruses, then its role in vivoas a nonspecific mechanism of resistance to virus disease would have tobe evaluated, particularly in relation to interferon-mediated interference.The latter is usually assumed to play a dominant role in most of thein vivo interference that has been described before and after thediscovery of interferon,

iT

19

LITERATtMJ.E CITED

I. McKinney, H.H. 1929. Mosaic disease in the Canary Islands, WestAfrica, and Gibraltar. j. Agr. Res. 39:557-578.

2. Hoskiris, X. 1935. A protective action of neurotropir, against-viscerotropic yellow fever virus in Macacus rhesus. Amer. J. Trop.Med. 15:675-680.

3. Isaacs, A.; Lindeman-n. J. 1957. Virus interference: I. Theinterferon. Proc. Roy. Soc. Ser. B 147:258-267.

4. Henle, W. 1950. Interference phenomena between animal viruses:A review. J. Immunol, 64:203-236.

" 5. Schlesinger, R.W. 1959. Interference between animal viruses, p. 157-1 89. In F.M. Burnet, and W.M. Stanley. (Ed.) The Viruses Vol. 3Academic Press, New York.

6. Finter, N.B. 1966. Interferons. W.B. Saunders Company, Philadelphia,', Pa. 3&0 p.

7. Huang, A.S.; Wagner, R.R. 1966. Defective T particles of vesicularstomatitis virus: II. Biologic role in homologous interference.Virology 30:173-181.

8. Hackett, A.J.; Schaeffer, F.L.; Madin, S.H. 1967. The separationof infectious and autointerfering particles in vesicular stomatitisvirus preparations. Virology 31:114-119.

9. Marcus, R.I.; Carver, D.H. 1967. Intrinsic interference: A new-- V type of viral interference. J. Virol. 1:334-343.

10. Cords, C.E.; Holland, J.J. 1964. Interference between enterovirusesand conditions affecting its reversal. Virology 22:226-234.

11. Pohjanpelto, R.; Cooper, R.D. 1965. Interference between poliovirusesinduced by strains that cannot multiply. Virology 25:350-357.

12. Roizman, B. 1965. Abortive infection of canine cells by herpessimplex virus: III. The interference of conditional lethal viruswith an extended host range mutant. Virology 27:113-117.

13. Berge, T.B.; Banks, I.S.; Tigertt, W.D. 1961. Attenuation ofVenezuelan equine encephalomyelitis virus by in vitro cultivationin guinea pig heart cells. Amer. J. Hyg. 73:209-218.

14. Zebovitz, E.; Brown, A. 1968. Pattern of viral ribonucleicacid synthesis in a temperature-sensitive mutant of eastern equineencephalitis virus. Bacteriol. Proc. p. 74.

&I

j Ii

20

S15. Brown, A. 1963. Differences in maximum and minimum plaque-formingtemperatures among selected group A arboviruses. Virology21:362-372. I

16. Zebovitz, E.; Brmwn, A. 1967. Temperature-sensitive steps in thebiosynthesis of Venezuelan equine encephalicis virus. J. Virol.1:128-134.

17. Zebovitz, E. 1965. A defined maintenance medium for supportingchick fibroblast monolayers and for plaque formation by Venezuelanand eastern equine encephalitis viruses. J. Infect. Dis.115:77-82.

18. Colon, J.I.; idoine, J.B. 1964. Factors affecting plaque formationby the infectious ribonucleic acid of the equine encephalitisviruses. J. Infect. Dis. 114:61-68.

19. Heller, E. 1963. Enhancement of chilcungunya virus replication andinhibition of interferon production by actinomycin D. Virology21:652-656.

20. Taylor, J. 1964. Inhibition of interferon action by actinomycin.Biochem. Biophys. Res. Commun. 14:447-451.

21. Bratt, M.A.; Rubin, H. 1967. Specific interference among strainsof Newcastle disease virus: I. Demonstration and measureiaent ofthe Interference. Virology 33:598-608.

22. Steck, F.T.; Rubin, H. 1966. The mechanism of interferencebetween an avian leukosis virus and Rous sarcoma virus: I.Establishment of interference. Virology 29:628-641.

23. Henderson, J.R.; Taylor, R.M. 1961. Studies on mechanismsof arthropod-borne virus interference in tissue culture. Virology13:477-484.

24. Allen, P.T. 1966. Replication ii- L cells of a substrain of westernequine encephalitis viz as exhibiting reduced virulence and theoccurrence of interference not mediated by interferon. Ph.D. Thesis,University of Texas.

SIt, I

f

I

• _____ ____________- - - - -- ___________________

7'

Ara I nr'z1;akiftad

*'t Detrick, Frederick, Mar..$, 22.701 AANON 'GROUP* A~ A. N)Vio a

ogene (McVI) Zebovira, , 0-A"II

Ocover 196e8 24

-( & Pftat Y WO. lort45Olt, Technical Fduscr-.p 477

10. MTOI~UTPINSTC49TQualif ed requester; mey obtcai copie n of this publication f he D nr.Foreign aneciounedent and disspenation of thia publication by DDC is not huegr±ri ed.

aelease or amoouncement to the puyelic ia oot authoruzed.

W; JIPPLVKMgTAR: NOT9u 12. t~~n~MILITARY ACTIVITYDepar~tment of tlt ArmyFort Detrick, Fredkrick, Marylar.d, 211701

13. AOS'WAACT

"'Itoerference among group A arbovirntees that did not involve thqre monestablishof interferu, Ai desevibed. lutterference was observed only if the interfortcngvLrus had an iedvantage s ver the challenge virus either in tine'or in multipliity

IIof l.fection. Adsorption, penetration, and utxcating of challenge virus Wor dotappear to be inhibited, but the gynthesis of infectious viral AM of the challengevirva ees significantly retarded. With temperature-adnaitive viruaes or mutante,[the repileation of vkzal RNA by the interfering virus was required to establish Iinterference, A mechanism of interference based on 4L c=mrtition for replicationsiteq or b uhatrates was compared with other possible explanations.

Lis. K.e, Words

Artcrerussgroup AVenezuelarn equine encer'1alitis virus

e8astern equine encephalitis vitus

4 Ribonucleic acid

~' ~ Unclassified