Isolation and characterization of recombinants between attenuated and virulent aphthovirus strains.

CopyrightC) 1987, AmericanSocietyfor Microbiology

Isolation and Characterization of Recombinants

between Attenuated

and

Virulent

Aphthovirus Strains

ANA T. GIRAUDO, ADRIAN SAGEDAHL, INGRID E. BERGMANN, JOSE L. LA TORRE,

AND EDUARDOA. SCODELLER*

Centro de Virologia Animal, Serrano 661, 1414 Capital Federal, Argentina Received 18 June 1986/Accepted 20 October 1986

Aguanidine-resistantmutantof the attenuated strain of aphthovirustype01strain Camposand theoriginal

wild-type strainwerecrossedto generaterecombinant viruses. Two independently derived recombinant viruses

were isolated. One isolate (RI) contained the P1 (structural proteins)generegionof attenuated strain and P3

(polymerase precursor) generegion ofthe wild-type strain. The other isolate (RII) had a genomicstructure

complementarytothat of RI, this is,P1 ofthe wild-type strain and P3ofthe attenuated virus. Recombinant

RIIinheritedsomein vitro phenotypic markers thatwerecharacteristic of theattenuatedstrain,whereas the

RIrecombinanthadinvitro behavior thatwassimilartothatof the wild-type strain. The data obtainedsuggest

that thepolymeraseprecursor(P3) oftheattenuated strain (01Campos)could be involved in the determination

of the attenuated phenotype for fetal bovine kidney cellsand,eventually, forcattle.

Although attenuated strains of polioviruses and aphtho-viruses (foot-and-mouth disease virus [FMDV]) have been

used for large-scale vaccination, up to few years ago little

was known about the molecular basis that determines the

attenuation processinpicornaviruses.

In recent years, however, work done by researchers in

several laboratories has allowed the accumulation of

consid-erable knowledgeonthe nature of the attenuation process in

poliovirus. Alterations locatedinthe capsid proteins of the

Sabin I strain have been suggested to play a major role in

determining the attenuated phenotype (1, 2, 3). Recently, a

recombinant poliovirus strain was constructed from

infec-tious cDNAclones ofneurovirulent (Mahoney) and

attenu-ated (Sabin I) strains. Analysis of the growth properties of

the recombinant viruses in tissue culture has allowed the

correlation of specific mutations that are located in the

genomicsegmentthatencodescapsid protein VP1and part

ofcapsid protein VP3 with in vitro phenotypic markers of

neurovirulence (14). Evidence has also been reported ofa

correlation between neurovirulence and asingle nucleotide

change in the 5' end ofthe noncoding region ofthe Sabin

type IIIpolio vaccinegenome (9).

Ithasalso been postulatedthatvariationsin the isoelectric

points of capsid proteinsVP1 andVP3between virulent and

lessvirulentstrains oftheTheiler murineencephalomyelitis

virus could be related to the differences in their degree of

virulence (22).

The involvement of capsid protein modifications in virus

attentuation has also been reported for other virus-host

systems(8, 18, 23, 24).

Inthecaseofaphthoviruses, littlework has been doneon

the mechanism(s) of attenuation. Harris and Brown (11)

havereported that in virus strain SAT1 limited changes in

thefingerprintof thegenomic RNA and ashortening ofthe

poly(C)-richtract areassociatedwith the processof

attenu-ation. Dawe and King (7) have reported a correlation

be-tweenalterationsin

capsid protein

VP1andmousevirulence

forthe FMDV-SAT1 strain.

Morerecently, wehave described

(21)

some biochemical

differences betweena strainof

aphthovirus

type01

Campos

*Correspondingauthor.

(O1C-O/E)attenuated for cattle and the wild-type strain(O1C)

from which it was derived. Changes in the viral structural

and nonstructural proteinsand in the lengthofthe

poly(C)-rich tract were found, but no correlation between the bio-chemical differences and the attenuated phenotype was

established(21).

To assesswhich regions of theO1C-O/Estrain are

respon-sible forthe attenuatedphenotype,weobtained recombinant

aphthovirus strains that carry different genomic regions of

thevirulentand attenuated strains.

Herewe report the biochemicalcharacterization and the

determination ofsomephenotypic markers,intissue culture,

oftwo such recombinants. It was found that recombinant

viruses that carry theP3/ABCD (P100)region of the genome

ofthe attenuated strain show in vitro phenotypic markers

similartothose of theparental attenuated strain.

MATERIALSANDMETHODS

Cells. Baby hamster kidney (BHK-21) cells were

culti-vated in Eagle minimum essential medium as described

previously (5).

Bovine kidney (BK) cells were obtained as primary

cul-tures from the Instituto Nacional de Tecnologia

Agro-pecuaria, BuenosAires, Argentina. Subsequentpassages(2

to 4) were performed in our laboratory with the same

medium as described above for BHK-21 cells, except that

fetal bovine serum (GIBCO Laboratories, Grand Island,

N.Y.)was used instead ofregular calfserum.

Virusstrains. Theoriginal01 Campos strainwasobtained

from tongue epithelium after bovine passages,

replicated

seventimes in BHK-21cells, and then plaque

purified;

one

clone (01C) was selected and was used

throughout

the

experiments.

The guanidine-resistant strain

(O1C-O/Egr)

was derived

from an attenuated strain ofthe FMDV 01

Campos

strain

(O1C-O/E)

(21) asfollows. The

01C-O/E

strain was

passaged

twice in BHK-21 cells in the presence of600,ugof

guanidine

hydrochloridepermlfor12to 24

h;

after the appearance of

cytopathic

effect bottleswerefrozen and thawed. This virus

stock was plated in the absence of

guanidine,

and several

plaques were isolated. The

guanidine-resistant

cloneswere

assayed by -titrationin the presenceorabsence of the

drug.

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TABLE 1. Plaque assay of the parent strains and theprogeny obtained by the yieldtestof the01C x 01C-O/Egrcrossunderrestrictive

and nonrestrictive conditions

Progeny virus titer(PFU/ml) in: Frequencyofpresumptive

Infecting viruses BHK-21 cells BK cells recombinants and

mutantsa Without guanidine Withguanidine Withoutguanidine Withguanidine

o1C 3.0 x 108 5.0 x 104 1.5 x 108 2.5 x 103 8 x 10-6

01C-O/Egr 2.5 x 108 1.5 x 108 1.0x 104 2.0 x 104 8 x 10-5

O1C x O1C-O/Egr 2.5 x 108 1.5 x 108 1.5 x 108 1.0 X 105 4 x 10-4

aRatio of infectious particles able to form plaques inBKcellsinthepresence of800,ugofguanidinehydrochlorideperml to totalinfectiousparticles.

Those clones showing the same titer under both conditions

wereselected.

Virus plaque titration. A total of 0.4 ml of serial 1:10

dilutions ofthe virus to be titrated was used to infect, in

duplicate, monolayersofBHK-21cells(5 x 106)orBKcells

(3 x 106) in glass bottles (21 cm2). Following adsorption

monolayers were overlaid with 6.0 ml of Eagle minimum

essential medium containing 40 mM HEPES

(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer

(pH 7.6) and1% karayagum(4). After 48 hofincubation at

37°C cellmonolayers were stained with0.2% crystal violet

andplaqueswerequantitated.

Clone isolation. The same procedure describedabove for

virustitrationwasusedfor cloneisolation,exceptthatin this

case karaya gum was replaced

by

1.2% agar

(Oxoid Ltd.,

London, England)and 2% fetal bovineserum wasaddedto

theoverlay. Plaqueswerevisualized aftermonolayerswere

stained withneutral red. Thecentersofwell-isolatedplaques

werepunched out to isolatethe virus.

Generation and isolation of recombinants. Two different

methods wereused togenerate and isolate recombinants.

(i)Conventional yield test. Monolayersof BHK-21 cells(5

x 106) in 21-cm2 flasks were coinfected with both parent

viruses at 10 PFU per cell. Control cultures were also

inoculated with either parent at the same multiplicity of

infection usedforthecoinfections. After 30 minof

adsorp-tion at 37°C, the unadsorbed virus was thoroughly washed

off with salineand maintenance mediumwas added.

The infected cultureswere incubatedat 37°Cfor 7h, and

at that time a marked cytopathic effect was seen. The

cultures were frozen and thawed once and clarified to

discardcell debris. Virus in each samplewasquantitated by

plaque titration under both restrictive and nonrestrictive

conditions (see below).

Virusfromthecoinfection thatwerecapable ofproducing

plaques under restrictive conditions was amplified by one

passage in BHK-21 cells, clonedtwiceundernonrestrictive

conditions, and amplified againin roller bottles.

(ii) Infectious center method. Theinfectious center method

was essentially that described by McCahon and Slade (17).

Plaques that were picked under restrictive conditions were

amplified in BHK-21 cells. From each of these isolations,

differentclones were obtained by plaque purification.

Labeling and preparation of cell extracts and of a

p56a-enriched fraction. Labeling with

[35S]methionine

and

immu-noprecipitation of viral-induced polypeptides in

BHK-21-infected cells, analysis of polypeptides on 10%

polyacrylamide gels, and isolation of viral

polymerase-enriched fraction were performed as described previously

(21).

Analysis of virion polypeptides by electrofocusing. Labeled

virionproteins that were obtained after acetone precipitation

ofpools ofpurified virions were suspended in

electrofocus-ing sample bufferand separated in nonequilibrium pH

gra-dients (NEPHGE) on slab gels (20 by 13 by 0.15 cm).

Electrofocusing conditions were carried outby the method

of O'Farrell et al. (19), as modified by King and Newman

(13).

Analysis of RNase Tl-resistant oligonucleotides by

one-dimensional gel electrophoresis. Preparations of 32P-labeled

RNA from the cytoplasm of infected cells, digestion with

RNaseTi, andelectrophoretic analysisof the digests were

performed asdescribedpreviously (15).

Kinetics of RNA synthesis. To determine the kinetics of

RNAsynthesis,24-well flat bottomdisposable plates(Nunc,

Roskilde,Denmark) containing5 x 105BHK-21cellsor2.5

x

105

BK cells per well were used. Monolayers were

infected with the viruses that were to be compared at an

inputmultiplicityof infection of 10 PFU/ml. After 30 min of

adsorption, the unadsorbed viruswasremoved and 0.4 ml of

mediumcontaining5 pLgofactinomycinD(SigmaChemical

Co., St. Louis, Mo.)wasadded. This mediumwasreplaced

after 30 minbymediumcontaining5 ,ug ofactinomycinD per

mlplus 15 ,uCiof[3H]uridine (30.6 Ci/mmol; NewEngland

Nuclear Corp., Boston, Mass.) per ml. Incorporation was

stopped byadding 1 mlof SDS-NET buffer(100mMNaCl,

50 mM Tris hydrochloride [pH 7.4], 1 mM EDTA, 0.05%

sodium dodecyl sulfate [SDS]) at the desired time. The

radioactivity of the trichloroacetic acid-insoluble material

wasdetermined. To evaluate thebackgroundofactinomycin

D-resistant nucleoside incorporation in BK cells,

mock-infected wells were assayed ateach time point.

RESULTS

Phenotypic properties of parental strains. To determine

appropriatemarkersfortheisolation and characterization of

recombinant strains, bothparental strains O1C andO1C-O/E

and a guanidine-resistant mutant derived from the latter

(O1C-O/Egr;

seeabove)weretitrated inBHK-21and BKcells

in the presenceorabsence of800 ,gof guanidine

hydrochlo-ride per ml.

Allthree strains grewequallywell in BHK-21 cells, which

is a permissive system for these viruses (Table 1). The

replication of strains O1C and O1C-O/E, however, was

stronglyinhibited by the presence of guanidine in the same

cells, whereas mutant O1C-O/Egr grew equally well in the

presenceor absenceofthe drug.

BKcells are a semipermissive system for the attenuated

virus strains; hence, in these cells only the wild-type strain

(O1C) reached titers similar to those obtained in BHK-21

cells, whereas strain O1C-O/E and the guanidine-resistant

mutantstrain derived from it(O1C-O/Egr)replicated poorly in

these cells. Strain O1C-O/Egr displayed its

guanidine-resistant phenotype,as wasthe casein BHK-21 cells. With

regard to plaque morphology, the wild-type strain (O1C)

elicited clear plaques that were ofsimilar size in both BK

andBHK-21cells(Fig. 1).InBHK-21 cells strainO1C-O/Egr

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produced plaques thatwereconsiderablylarger than thoseof

strain 01C; however, the border of the plaques were not

clear, as was the case in the wild-type strain. Plaques

produced by 01C-0/Egr in BK cells were small and ill

defined. Identical plaque featureswerealsodisplayed bythe

guanidine-sensitive, attenuated strain 01C-O/E (data not

shown).

Generation and isolation of recombinantviruses. Based on

the phenotypic features of strains 01C and 01C-O/Egr, we

usedasselective markersthe ability of 01C-0/Egrtogrowin

thepresenceof 800 ,ug of guanidine hydrochlorideperml and

thecapacity of strain01Ctogrow athigh titers in BK cells.

The recombination experiments were carried out as

de-scribedaboveby usingasrestrictive conditions theplating of

progeny virus in BK cells in the presence of 800 ,ug of

guanidine hydrochloride per ml. The data obtained on

titration ofprogeny produced by the yield testaregiven in

Table 1. The proportion of plaques developed under

restric-tive conditions was fivefold higher in the progeny from

mixed infection than in theprogenyobtained frominfection

with 01C-0/Egr alone (recombination frequency, ca. 4 x

10-4).

The10plaques thatwereformed under the conditions

described above by the progeny of mixed infection were

picked up, amplified as described earlier, and investigated

with respect to their resistance to guanidine and to their

capacity togrowinBK cells. All 10 cloneswere guanidine

resistant; and 7 of them exhibited high titers in BHK-21 or

BK cells,as wasexpected for recombinants.

The other three clones replicated in BK cells at titers

similartothat of strain01C-0/Egr; this observation could be

an indication that these clones are truly parental strains.

Further biochemical analysis of these viruses supported this

assumption. The seven clones with the phenotypic

charac-teristicsexpected for recombinant viruseswere selected for

biochemical characterization to assess whether they were

truerecombinants (see below).

BHK BK BHK BK

ofc O,C-O/Egr

BHK BK BHK BK

RI

RII

FIG. 1. Representativeplaque morphologyofparentand

[image:3.612.313.554.95.170.2]

recom-binant strainsonBHK-21 and BK cells.

TABLE 2. Growthcharacteristicsof recombinants RI and RIIin BHK-21andBKcellsa

Titers(PFU/ml) in thefollowing

Cell recombinants:

RI RII

BHK-21 2.0 x 107 2.0 x 108

BHK-21with guanidineb 2.0 x 107 1.0 x 108

BK 2.0 x 107 5.0x105

BKwithguanidineb 2.0x 107 2.5 x 106

a The virus plaque titration assay used was the karaya gum overlay method.

bGuanidine(800 ,ug/ml) was included in theoverlay medium.

Alternatively, recombinants were obtained by using the infectious center assay reported by McCahon and Slade (17).

By thisprocedure five plaques were isolated under

restric-tive conditions, and viruses recovered from each isolation

were amplified in BHK-21 cells. Biochemical analysis of

viruses recovered from four of the plaques indicated that

they were indistinguishable from the parental strains,

whereas viruses recovered from a fifth plaque elicited the

biochemical characteristics of recombinants viruses (see

below). It is well known that this methodproduces mixed

populations of viruses rather than single homogeneous

strains(17);therefore,recombinants recovered from the fifth

plaque were cloned in BHK-21 cells, and several clones

were recovered. Viruses from two of the plaques were

guanidine resistant and elicited titers of105 PFU/ml in BK

cells. Although these features are quite similar to those of

the01C-0/Egr strain, the plaques produced by these clones

in BK cells were well defined and small; hence, they were

clearly distinguishable from the typical ill-defined, small

plaques produced by the 01C-O/Egr parent strain in this

system. Therefore, these clones wereselected for

biochem-ical characterization.

Representative clones derived from both recombination

methods were titrated in BHK-21 and BK cells.

Recombi-nant I (RI) is a representativeof the presumptive

recombi-nantclones selected in theyieldexperiment,whereas

recom-binant II (RII) is a clone that was selected by using the

infectious center assay. RI elicited a high titer in both

cellular systems(Table 2),as wasexpectedforastrain(O1C)

with a phenotype like that of the wild type; on the other

hand, the titer of RII in BK cellswas ca. 3 log units lower

than that in BHK-21cells. Thisbehavior is similartothat of

the attenuated parent strain (01C-0/Egr).

Thefeatures of theplaques induced byboth recombinants

(RIandRII) in BKandBHK-21cellsareshown inFig. 1. In

BHK-21cells RIproduces plaquesthatareindistinguishable

from those produced by strain

01C-0/Egr,

whereas the

plaques producedin BKcells, althoughsmaller than those of

BHK-21 cells, show a similar morphology. On the other

hand, in BHK-21 cells RII produces plaques that were

indistinguishable from those of strain 01C, but in BK cells

RII elicited very small

(pinpoint)

clearplaques. Therefore,

taking into account differences between titers in BK and

BHK-21cells, RIwould bethoughttohaveawildtype-like

behavior. On the contrary RII showed an attenuatedlike

behavior,

according

tothetiters obtained in BK and BHK-21

cells.

Biochemicalanalysis ofputative recombinants. To screen

theputative

recombinants,

weusedasnonselective markers

biochemical differences between strains 01C-0/E and 01C

which mapped on the viral

capsid (P1)

and

polymerase

precursor(P3)

regions

ofthe viral genome

(21).

In

addition,

,,*

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(i 4.

0' 0 ~0

0-K

Ki

t.

' ok

o0?

-ft

=VP3

alCs

-

* -

i_ _ s

VP,

FIG. 2. Analysisofviral structuralproteins ofparentand recom-binant virusesbyNEPHGE. Forstraindesignations, see text.

thepoly(C)-richtract,which is locatednearthe5' endof the

viral genome of the attenuated strains, is considerably

shorterthan that of thewild-type strain from which it was

derived. Although thesetraitshave beendefinedby

compar-ing 01C with 01C-0/E, these differences in P1, P3, and the

poly(C) sequences were maintained when 01C was

com-pared with01C-0/Egr(data notshown).

From the 10 clones obtained by theyield method, 3 were

phenotypically andbiochemically indistinguishable fromthe

parent strain01C-0/Egr, whereas the other 7clones elicited

phenotypesthat were predictedfor recombinant viruses.

Biochemical analysis of the 7 putative recombinants

showed that all of them had identicalcapsidprotein

compo-sitioninNEPHGE andSDS-polyacrylamidegel

electropho-resis (data not shown). One of the recombinants (RI) was

&i\4' C'

K0 _(p00

l0'

,0_'1>

'N,1

K

an- --Am _ _ m Pco

- - -- P88

a

-_-- a U p72

selected as being representative of this group and was

analyzed inparallel with RII and the parent strains.

The pattern of structural (capsid) proteins of the viruses

analyzed by NEPHGE (see above) is shown in Fig. 2. The

migrationpattern of VP1-VP2 and VP3 of RI is

indistinguish-able from that of the attenuated parent (01C-0/Egr) and is

clearly different from the pattern ofproteins elicitedby the

wild-type parent strain 01C. In the case ofRII, which was

obtained by the infectious center assay, the pattern of

structuralpolypeptides is indistinguishable from that of the

wild-type strain but clearly different from that of the

atten-uated parent.

Analysis of virus-induced polypeptides (see methods) by

SDS-polyacrylamide gel electrophoresis shows that RI

ac-quired the polymerase precursor P3 (P100) that is

character-istic of the wild-type strain, whereas RII inherited P3 of its

attenuated parent (Fig. 3).

Analysis of p56a of theparental strains and both

recom-binants by NEPHGE (21) shows that RII inherited an RNA

polymerase polypeptide that was indistinguishable from that

of theattenuated parental strain01C-0/Egr, whereasp56a of

RI was similar to that of the wild-type strain 01C (Fig. 4).

Not all of the recombinant viruses recovered showed this

trait, however. Two of those clones that carry RNA

poly-merases thatresembled neither parent are shown in Fig. 4.

As suggested by McCahonetal. (16), thesevariations could

be due to recombinationevent(s) within the codingregion of

that protein.

The results shown in Fig. 2 to 4 demonstrate that both

strains that were analyzed in this study are true

recombi-nants. RI carries the P1 region of the genome of the

attenuatedparent and the P3region of the wild-type strain,

whereas RIIcontains the reverseofRI, in that it carries P1

of the wild-type strain and P3 of the attenuated parent.

Further evidence was obtained by genomic analysis ofRI

and RII and their respectiveparents by RNase Ti maps on

one-dimensional gels. It was found that RI carries theshort

poly(C)-rich tract that is characteristic of the attenuated

strain (21), whereas RII inherited the poly(C)-rich tract of

thewild-typestrain(datanotshown). These results suggest

that besides the P1 region, RI possibly carries all the

Ko°so 0°'s 2

_C,

}0S°

_{C, x}0'

0 KpI

4qmw

M

q11qpp1

p am p56a

.

'- - - _ p56a

__m mm -_ VPo

______*m mm.e

1 40_

q&MW4_a

]JPI-3

_a _ _ _

FIG. 3. Polyacrylamide gel electrophoresis of virus-induced

polypeptides in BHK-21 cells infectedwith parental and

recombi-nant viruses; monolayers were pulse-labeled, and viral-specific

proteinswereisolated and analyzedasdescribedin thetext.

FIG. 4. Analysisofp56aof recombinants and parental strains by NEPHGE. RIais a recombinant variant representing one of the

seven clones obtained in the yield assay. Rlla is a recombinant variant recovered by further plaque purification of the original isolate obtainedbytheinfectiouscenter assay (see the text).

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sequences upstreamof theP1region or at least up to the 5'

endofthe poly(C)-rich tract of the attenuated strain.

Viral RNA synthesis in BHK-21 and BK cells. Strain

01C-0/Egr and its parent strain 01C-0/E are deficient in RNA

synthesis in cells from bovine origin because they reach at

most 10% ofthe level of RNA synthesis obtained in strain

01C (data not shown). Therefore, we investigated whether

this trait was inherited by RI or RII. The kinetics of RNA

synthesis werestudiedin BHK-21 and BK cells. The

kinet-ics of RNA synthesis induced in BHK-21 cells by the

parental, RI, and RII strains are shown in Fig. SA. The

attenuated strain 01C-0/Egr reached roughly 50% of the

levelsachieved in thewild-typestrain, which wasthe same

as that achieved in its parental strain 01C-0/E (data not

shown). RIandRIIdiffer greatlyin the synthesis of specific

viral RNA induced in BHK-21-infected cells. RI reached

synthesis rates

similar

to those reached in the attenuated

strain,whereas RIIelicitedan RNAsynthesisratesimilarto

that ofthe wild-type strain.

Ontheother hand,in BKcells(Fig. SB)thehighestvalues

were reached by the wild-type strain, and as stated above,

01C-0/Egr reached values that were 10% ofthose obtained

with strain 01C. The rate ofRNA synthesis in BHK cells,

which was highest in RII, fell dramatically in BK cells,

showing values similar to those of01C-0/Egr. The rate of

RNA synthesis of RI increased considerably in BK cells,

showing RNA synthesis rates similarto those ofthe

wild-typestrain. These results suggest that thefailureto

synthe-sizeRNAinBKcellswasinheritedbyRIIratherthanby RI,

in spite ofthefact that synthesis in BHK-21 did notreflect

thisfact.

The rate ofRNA synthesis in BK cells was also

estab-lished for other recombinants

(RIa

and RIIa; Fig. 4) that

carrypolymeraseswithchargesthat aredifferentcompared

with their parental strains. It was found that RIa has a

slightly lower rate of RNA synthesis than RI, whereas

recombinantRllareached values slightly higherthan those

ofRII(datanotshown).No otherbiochemicalor

biological

differencesbetween RI-RII andRIa-RIIa,

respectively,

were

detected. Therefore, it seems that theonly difference found

between viruses thatcarry

parental

orunique p56awasthat

ofadifferentrateofRNA

synthesis

in BK cells.

DISCUSSION

Wehave

recently

describedthebiochemical

characteriza-tion of a host range mutant of FMDV serotype 01 strain

Campos

thatwasattenuatedfor cattleand thatwasobtained

throughadaptationonserialpassagesofthe

wild-type

strain

in chickenembryos. Theattenuated strain showeda

short-enedpoly(C)-richtractandseveralchangesin structural and

nonstructuralproteins.Theinvolvement ofthosechangesin

the determination of the attenuated

phenotype, however,

was notestablished.

Recently, it has been demonstrated (12, 16) that in

aphthovirus intra- and

intertypic reconmbinants

can be

ob-tained at

high

frequencies

in the

laboratory.

Therefore,

recombinants between

wild-type

andattenuated strainswere

constructed to ascertain which parts of the attenuated

genomewererelevant forattenuation. Of 10 clones isolated

by

the

yield method,

7were consideredtobetrue

recombi-nantsbecause

they

containedtheP1

region

ofthe

01C-0/Egr

genome and the P3

region

ofthe01C genome. The fact that

virusesofthe RIgroup inherited the

poly(C)-rich

tractof the

attenuated strain is additional evidence that supports the

idea that these viruses are truerecombinants.

A

10r

8-IV

0.

U

.-.OAc

oOA,-O/Egr h- RI

Mo

Rck

*--a Mock

B

HOURS AFTER INFECTION

FIG. 5. Kinetics ofRNAsynthesisof recombinants andparental

viruses in BHK-21 (A)and BK(B)cells. Fordetails,seethetext.

Basedonbiochemicaland

biological

data

presented here,

itcanbe

postulated

that attenuatedviruses inwhichtheP3

regions

have been

replaced by

an

equivalent region

ofthe

wild-type

strain

(O1C)

have also been relieved of its

con-straint(s)

togrowinBKcells.

Based onthe restrictive conditions used for

selection,

it

was

expected

that

only

those recombinantswith the

capacity

togrowat

high

titers inBKcells would beisolated.

By

using

the infectious center assay,

however,

a recombinant

(RII)

withalimited

capacity

togrowin BK cells

(like

the

attenu-ated

strain)

wasisolated. Thiswas not

surprising,

consider-ing

the differences between the methods usedtoobtainthe

recombinants. In the

regular yield

assay, the viruses

pro-duced in BHK-21 cells were

plated

in BK cells

plus

guanidine,

sothat

plaques

were

produced by

a

single

infec-tive virion. On the other

hand,

in the

infectious

centerassay

BHK-21 infected cells were

plated

1 h after

adsorption

on

the BKcell

monolayer,

and

plaques

were

produced by

the

progenyoftheseinfectedBHK-21cells.

By

thenatureof this

method,

itis

likely

thatmore thanone recombinantcanbe

found in the same

plaque (17).

Ifrecombinant RI and RII

were

produced

in the same

cell,

the

original

plaque

from

which the

virus(es)

was recovered would contain both

recombinants.

Taking

intoaccountthatthe

virus(es)

in this

plaque

was

amplified

in BHK-21 cells

before

itwascloned

and that recombinants ofthe RII type reached a titer that

was 10timeshigher than that in RI in these cells

(Table 2),

it

is understandable that clone RII can be

preferentially

se-lectedafter

cloning.

Biochemical

analysis

oftherecombinantobtained

by

this

method demonstrated

that,

indeed,

RII carried P1 and the

poly(C)-rich

tractofstrain

01C

and P3ofstrain

01C-0/Egr.

With

regard

toother

general

characteristics ofthe

recombi-nants,itwasfound that RI grew

equally

wellin BHK-21 and

BK cells

(Table 2),

produced

large-sized plaques

in both

systems, andwasabletoinduceanRNA

synthesis

level in

BKcells similartothatofthe

wild

type.On the other

hand,

thetiter of RII in BHK-21 cells was3

log

units

higher

than

that in BK

cells,

but in this

system

the recombinant elicited

verysmall

(pinpoint) plaques,

and therateof RNA

synthesis

reflectedthatofits attenuatedparent

01C-O/Egr.

Taking

all

this into account, one could assumethat RII shares the in

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http://jvi.asm.org/

[image:5.612.308.552.69.259.2]

vitro markers of attenuation of strain

01C-0/Egr.

We have

previously postulated (21)

that theincreased

electrophoretic

mobility

ofP100 could be duetothe

deletion(s)

that

develops

during

the processof

attenuation; therefore,

it is

tempting

to

speculate

that those

putative

deleted

regions

could be

in-volved in thedetermination ofthe attenuated

phenotype.

Ifattenuation lies outside of the

capsid proteins,

then it

would be feasible

by

means of recombination to introduce

the

noncapsid

part ofthe genome of an attenuated donor

strain,

which is

responsible

forthe attenuated

phenotype,

in

newemergent variants without

altering

the

antigenic

struc-tureofthesenewstrains. Thetime

required

todothiswould

be

considerably

shorter than the time necessaryto

develop

anattenuated virus

by

theclassical

procedures. Approaches

of this type have beenshowntobeuseful in the construction

of new attenuated strains in influenza virus

(25).

We are

currently

testing

this

hypothesis using intertypic

recombina-tion.

Major

determinants ofattenuation were located in the 5'

halfof the viral genome,

specifically,

in the

capsid region

of

Sabin type I

(1, 2, 3)

andin the

5'-noncoding region

ofSabin

type III

(9).

Recently, however,

Omata et al.

(20)

have

shownthat

multiple

determinantsofattenuation reside in the

poliovirus

Sabintype I genomeatlocations other than in the

capsid region.

Evidence forothervirus families also existsthat

points

the

fact that structural

proteins

are

important

determinants of

virulence

(8,

18, 22, 23, 24).

In this

study

a

noncapsidal region

of the viral genome

appeared

to

play

a

major

role in

determining

the in vitro

phenotypic

markers of attenuation.

It appears,

however,

that with the in vitro markers of

attenuation that are inherited

together

with

P3, plaque

fea-tures appear to be

mainly

linked to the inheritance of P1

(compare

the

plaques

of recombinants and

parental

strains in

Fig.

1).

Although

it has been shown that BK cells are a

good

system for

testing

viruses thatareattenuatedforcattle and

that all theknownattenuatedviruses reflecttheir features in

this cell

(6, 10;

Pablo

Auge

de

Mello, personal

communica-tion),

we must

keep

in mind that the definitive

proof

of

attenuation will be

provided by

resultsof

pathogenicity

tests

in

cattle,

whichare

currently

in progress.

ACKNOWLEDGMENTS

Wethank A. M. Sadir andA. Zabal(INTA) forprovidingPBK cells and C. Ricarte and E. Yampolsky for excellent technical assistance.

This workwassupported by ConsejoNacional deInvestigaciones

Cientificas y Tecnicas and by Instituto Cientifico Paul Hermanos

(Bs. As., Argentina). I.E.B. isarecipientofaHumboltFellowship.

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