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0022-538X/85/060690-07$02.00/0

Copyright ©D1985,American SocietyforMicrobiology

Genomic

Expansion of

Marek's Disease

Virus

DNA

Is Associated

with

Serial

In

Vitro Passage

ROBERT F. SILVA* AND RICHARD L. WITTER

U.S. Department ofAgriculture, Agricultural Research Service, Regional Poultry Research Laboratory, East Lansing, Michigan 48823

Received28December 1984/Accepted11 March 1985

AnEcoRI restriction endonuclease pattern of Mdll virus DNA, a very virulent strain of Marek's disease

virus (MDV), was obtained by using total cellular DNA from infected cells. With the EcoRI restriction

endonuclease pattern and a published BamHI map of MDV (Fukuchi et al., J. Virol. 51:102-109), we

constructeda partial EcoRImapofa series of MDV clones (gift from H. J. Kung). The cloneswereusedto

identifyaregion of the Mdllgenomewhich isalteredastheoncogenic virus is passagedinvitro. This region wasmappedintoa1.8-kilobasesegmentintheinverted-repeatsequencesflankingthe longunique region ofthe

virusgenome.Thealterationappearedtoresultfrom multiple DNAinsertionsthatproducedanincreaseof 0.6

to5.4kilobases. Although theexpansion of this regiondid notdiminishtheabilityof MDVtoreplicateinvitro, itmaybeassociated with the loss of Marek'sdiseaseoncogenicity.

Marek's disease virus (MDV) is an oncogenic herpesvirus

of chickens that can be attenuated by repeated in vitro passage (2, 9, 14). Several reports have shown that the

BamHI restriction endonuclease (RE)pattern ofthe attenu-atedvirus lacks fragments present in the oncogenic parent (6, 7, 12). More recently, the RE patterns of the virulent

BC-1 andJM strains ofMDV were comparedtoattenuated

MDV. The alteredregion resulted fromthe rearrangementof

viral DNA during serial passage and mapped to BamHI-D and -H located in the inverted repeats of the genome (K.

Hirai, K. Ituka, N. Nakajima, and S. Kato, Abstr., Int. Symp. Marek'sDisease,1984, p. 14). Fukuchiand

co-work-ersalso reported findingalterations and expansion ofDNA inBamHI-D and -Hofvirulent GAand JM virusesfollowing serialpassage(K.Fukuchi,A.Tanake,M.Suto,J.Donovan,

L.Eklund,J.Jessip, and M.Nonoyama,Abstr.,Int. Symp.

Marek's Disease, 1984, p. 16).

We have investigated Mdll virus, classified as a very

virulent strain of MDV, and several high-passage

prepara-tionsof this strain which havelost theironcogenic

potential

(14-16).Weanalyzedthe RE patternsof Mdll viralDNAto

identify regions ofthe MDV genome which are associated withoncogenicity. Traditionally,the REanalysis ofMarek's

disease virusDNA,aswellasother virus systems, hasbeen

performed on DNA isolated from cell-free virions. How-ever, the strongly cell-associated nature of MDV makes

isolating viral DNA difficult. We report there that it is

possible

to obtain accurate RE patterns of viral DNA by using totalDNAfrom MDV-infectedcells. By this

simplified

procedure, we found that EcoRI fragment F present in

oncogenicMdll viruswasnotpresent inREdigestsofDNA from cells infected with attenuated Mdll. Fragment F

disappeared as a result of the heterogeneous expansion of DNA within the inverted repeats flanking the long unique region of the Mdll genome.

MATERIALS AND METHODS

Viruses andcells. Primary duckembryo fibroblast (DEF)

andchickenembryofibroblast (CEF)cultureswereprepared

as previously described (13). Semiconfluent DEF or CEF

* Correspondingauthor.

cultureswereinfectedwithcell-associated MDVata multi-plicity of infection ofapproximately 0.2 and maintained in F10-199medium, supplemented with 1% calfserum. When the cultures displayed extensive cytopathic effects (CPE),

the infected cells were transferred to fresh DEF or CEF cultures. The Mdll strain of MDV retained its virulence after10 passagesin DEF(Md11/10). Mdll virus, provision-ally classified as a very virulent MDV (15), was serially passagedin vitro. Alloftheserially propagated viruseswere

capable ofprotecting chickens againstachallenge by path-ogenic MDV. In addition, none of the serially propagated

viruses induced gross Marek's disease lesions. However,

I

FIG. 1. RE patterns ofpurified MDV DNA and MDV-infected cell DNA. Total DNA from DEF or purified virus DNA was digestedwithEcoRI, separated ina0.6%agarosegel,and stained with ethidium bromide. Lanes: 1, DNAfrom uninfected DEF; 2, DNA from MDV-infected DEF; 3, DNA extracted from purified MDV virions.

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GENOMIC EXPANSION OF MDV 691

F

DIE H

B

w

FIG. 2. EcoRI RE digestion of Md11/10 DNA. DNA was ex-tractedfrom Mdll/10-infected DEF, digested with EcoRI, separated in a 0.6% agarose gel, and stained with ethidium bromide. A photograph, taken with UV illumination, was scanned with a densitometer.

Mdl1/10

Mdl1/60

Mdl1/81

Mdllvirus at passage 57producedtypical Marek's disease-typemicroscopicnervelesions when inoculated into day-old

chicks. Nonervelesionswerefound in birds inoculatedwith Mdllvirusesatinvitropassagelevels of 75, 90, and100(14, 16). Beforethe currentstudy,theseviruseswerepassagedin

vitro an additional three to six times and have been

desig-natedMdll/60, Mdll/81, Md1l/93, andMdll/103.

Preparation of DNA. ToisolateDNAfromMDV-infected DEFandCEF, monolayercultures showing extensive CPE were washed with cold Dulbecco modified

phosphate-buff-ered saline, scraped offtheplates, andincubated for 4 hat

45°C in 10 volumes ofNES-proteinase K solution (0.15 M

NaCl, 0.1 M EDTA, 1% sodium dodecyl sulfate [SDS],

proteinase K at 100 ,ug/ml). The DNA was extracted twice with an equal volumeofphenol-chloroform-isoamylalcohol (25:24:1) and once with chloroform.

PlasmidclonescontainingEcoRI restrictionenzyme

frag-mentsof GA/22 DNAwere agiftfrom H. J. Kung and have

TABLE 1. EcoRI REfragments from Md11/10'

Fragment Size (kb)

A... 14.5

B... 12.5

C... 11.8

D... 7.8

E... 7.8

F... 6.9

G... 6.1

H... 5.7

1.

... 5.15

J... 4.4

K... 4.1

L... 3.35

M... 3.08

N... 2.8

O... 2.5

P... 2.3

Q... 2.2

R... 2.0

S... 1.9

T... 1.7

U... 1.6

V... 1.4

W... 1.35

X ... ... ... 0.8

"Total cellular DNA from Mdll/10-infected DEFwas isolated, cut with

EcoRI. separatedon a0.6%agarosegel,andstainedwithethidium bromide as describedinthe text.

FIG. 3. RE pattern comparisonofoncogenic MDV and

attenu-ated MDV. DNAwasextracted fromMdll-infectedcells, digested with EcoRI, and processed as described in the legend to Fig. 2. Mdll/10, Oncogenic virus at tissue culture passage 10; Mdll/60, nononcogenic virusatpassage60; Mdll/81, nononcogenicvirusat

passage81; Mdll/93,nononcogenicvirusatpassage93;Mdll/103, nononcogenic virusatpassage103.

been described (4). Growth of the clones and extraction of DNA followed established procedures (8). MDV DNA, purified from cell-free virions, was a gift from L. Lee

(Regional Poultry Research Laboratory, East Lansing, Mich.).

By standard techniques, 32P-labeled DNA probe was

prepared by nicktranslating 200 ng of DNA for90 min at 13°C with [ot-32P]dCTP (NewEnglandNuclearCorp.) (8).

RE digestion and gel electrophoresis. RE enzymes were

purchased from either Bethesda Research Laboratories or

New England BioLabs and used in agreement with the specificationsof the manufacturer. Foragarosegels, 1to 10 ,ug of RE-cut DNA perlanewaselectrophoresed overnight

at<2 V/cm in0.6%agarosegelsin TBEbuffer(89mMboric acid,2mMEDTA, 89 mM Tris-borate[pH 8.0]).Gels were

stainedfor45minin0.5 ,ugof ethidium bromideperml and photographed under UV illumination. The size of the RE-generated fragments was estimated by coelectrophoresis

withaHindIll digest of lambdaphage DNA.

DNAtransfer andhybridization.Theagarosegels, contain-Mdl1/93

Mdl1/103

VOL. 54,1985

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692 SILVA AND WITTER

0 20 40 60 80 100 120 140 160 180

a a a a ..kI .a a I a a . .a a I

BamnH

H

i

.

1

..N

.Ii

i.I

.

H...

L I D G C F KKI E I J B KN I A

BgIl

! ' "

i

I

i

H

i i

i

i

H

*

J F I G A I B K D E C H F J C C

F I A E b C HJ C

EcoRI

IHHHIIII4

QF K I G TMN

F KK B K

I-1HWO NH

E L1L2D C B XFQ 0 1

ITRI RS

FIG. 4. Partial EcoRI map of MDV clones. DNA from the pBR328 clones of GA virus was used to probe BamHI-, BgIl-, and SmaI-digested DNA from MDV-infected DEF,asdescribed in thetext.The EcoRImap wascreated by comparing the resulting Southern blot

patternswith published BamHI, BglI, and SmaImapsof MDV (3). The scale indicates kb of DNA.

ing the separated RE-digested DNA,weresoaked for 30min

in 0.2 M NaOH-0.5 M NaCl and neutralized by soaking three times (10min each time) in TAE buffer (20 mM sodium acetate, 1 mMEDTA, 40 mM Tris base [pH 7.4]). The DNA

was electrophoretically transferred to Zeta-Probe

mem-branes (Bio-Rad Laboratories) in a Bio-Rad Trans-Blot

systemwithTAEbufferat20 V for 30min, followed by 30 V for3 h. The membraneswerewashed briefly in TAE buffer,

dried, and baked for 2 h at80°C.

Forhybridizations, the membraneswereprehybridized in

heat-sealed plastic bags for 2 h or more at 42°C in 47% deionized formamide-5x Denhardtsolution(100xDenhardt

solution consists of 2%Ficoll, 2% polyvinylpyrrolidone, 2% bovineserumalbumin)-5x SSPE (20x SSPE consists of 3.6

MNaCl, 20 mM EDTA, 200 mM NaH2PO4 [pH 7.4])-250 ,ug/ml of sheared salmon sperm DNA-10% dextran

sul-fate-0.1% SDS. The membraneswerehybridizedat42°C for

over 3

Cot1/2

in fresh hybridization buffer containing the

denatured 32P-labeled DNA probe at approximately 1.5 x

106 cpm/ml of buffer.

Filterswerewashed in 2x SSC (20x SSC consists of 3 M

NaCl, 0.3 M sodium citrate [pH 7.0])-0.5% SDS for 5min,

followedbya15-min wash in 2x SSC-0.1% SDS. The filters werethen washed in 0.1x SSC-0.5% SDS at68°C for 2 h,

o;a.

DNALfro en.F

'O~~~~~~~~~~~~~~~~~~~~''

EKoRI-digested2DNA

....;...E

described in the text. The abscissarepresentstheleft-to-right migrationof REfragments.Theordinateindicatesrelative absorbance. Scans

Aand Bwere made fromadjacent lanes of thesame autoradiogram.A,Md11/10DNA;B Md11/81 DNA.

Smal

I I . .. . m . I 9 .. .. .. .. ..

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GENOMIC EXPANSION OF MDV 693

F

fl

FIG. 6. EcoRI-F probe ofBglI-digested DNA from oncogenic and attenuated Mdll. 32P-labeled EcoRI fragment F was used to probe BglI-digested DNA from Mdll/10- and Mdll/81-infected DEF.Theresulting autoradiogramwasscanned witha

microdensi-tometer,asdescribed in thelegendtoFig. 5. A,Mdll/10DNA; B, Mdll/81 DNA.

followedby a30-minwashat68°Cin fresh0.1x SSC-0.5% SDS. The filters were air dried and exposed to X-ray film (KodakX-Omat AR)at -70°C. For some experiments, the autoradiograms and films were scanned with a microdensi-tometer(GS 300, Hoefer ScientificInstruments).

RESULTS

Comparison of the RE patterns of viral DNA and total cellular DNA. We haveconsistentlyseenethidium bromide-stained DNA bands in agarose gels during RE enzyme

analysisof DNA from infected DEF. These bands could be MDV DNA since productively infected CEF can have in excessof150copies of the MDVgenome percell(10).That these fragments were MDV specific is shown inFig. 1. No bandsare seenin theEcoRI-cut DNA from uninfected DEF

(lane 1). However,the EcoRI RE band pattern obtained with DNA from MDV-infected DEF (lane 2) is identical to the EcoRI pattern of purified viral DNA (lane 3). Only the high-ethidium bromide-staining backgroundof cellular DNA distinguishesthelanecontainingtotal infected cellular DNA from the lane withpurified viral DNA. Similar results were

obtainedwithBglI, SmaI,andBamHI REenzymedigestsof

DNAfrom MDV-infected DEF and CEF(datanotshown). Becauseof the

difficulty

of

isolating

cell-free viral

DNA,

all further RE

analysis

was

performed

withtotal cellularDNA

from MDV-infectedDEFandCEF.

Alterations in the DNA of high-passage MDV. We have observed that when theoncogenicMdll virus is

repeatedly

passaged in

vitro,

the virus loses its

pathogenicity (14, 16).

We wereinterested in

determining

whetherwecould detect differences in theREpattern of low- and

high-passage

Mdll virusthatcould becorrelated with the loss of

oncogenicity.

A microdensitometer

tracing

of the EcoRI

fragments

ob-tainedwith the

oncogenic, low-passage

Md11/10 isshownin

Fig.

2. Fragment sizes are listed in Table 1. An identical

EcoRIpatternwasobtained with the

oncogenic

GA/22 virus (data not

shown).

The sum ofthe

fragment

sizes was 117

kilobases

(kb)

and was

certainly

an underestimate since several bands

appeared

tobepresentin greaterthan molar

quantity. Unfortunately,

the

background

smear of DEF DNA made

estimating

accurate molar ratios very difficult. Basedon

peak

height,

we

judged

severalEcoRI

fragments

to be present in greaterthan molar

quantities. Fragments

that have

consistently appeared

to be present in greater than

molar

quantities

include

EcoRI-F, -H, -L, -T,

and -W. A moreaccurate estimateofthe size ofthe MDV genome is around180 kb(3).

Wefound

striking

differenceswhenwe

compared

the RE patterns of

high-passage,

nononcogenic

Mdll virus DNA with the REpattern of low-passage,

oncogenic

Mdll virus

DNA. A

portion

ofthe EcoRIRE patternobtainedfrom the

oncogenic

Mdll virus after tissue culture passage 10 is

shownin

Fig.

3

(top).

The

remaining

RE

tracings

displayed

EcoRI-cut DNA from

nononcogenic

Mdll viruses after

passages60,

81, 93,

and 103. EcoRI

fragment

F

appeared

to

be present in greater than molaramounts in Md11/10 virus

and was absent from the

high-passage

MDV viruses. In

addition,

a few faint bands

appeared

between the C and D

fragments

that maynothavebeen present in

Md11/10

virus

DNA. The loss of the EcoRI-F and the appearance of

fragments

between EcoRI-C and -D were the

only

altera-tionsweobserved inthe EcoRI patternsofanyofthe

Mdll

passaged

viruses. To further

analyze

the

disappearance

of

the F

fragment,

wedecidedtomap EcoRI-F andto

probe

the RE

digests

ofMdll with the

32P-labeled

EcoRI clones. The first stepwas tomap the recombinantDNAclones.

Partial EcoRI map of MDV. On the basis of

size,

we

determined that at least 19 of the MDV EcoRI

fragments

were containedin the

pBR328

clones. To mapthese

clones,

we

digested

DNA from MDV-infected DEF with

BamHI,

BgIl,

or

SmaI,

and the

digests

were

separated

on a0.6%

agarose

gel.

The

fragments

were transferred to Zeta-Probe

membranesasdescribed above and

individually probed

with the 19 clones. The

BamHI, SmaI,

and

BglI

fragments

that weobservedin theSouthernblotswereidentified

by

match-ing

them with the

fragments reported by

Fukuchietal.

(3).

Weused their detailed

BamHI, BglI,

andSmaIREmapsof

MDVto construct a

partial

EcoRI map. In several cases,it

waspossibletoonly roughly mapthelocation oftheEcoRI

clones. For

example,

the order and exact locations of EcoRI-O and EcoRI-I could not be determined

precisely.

Our data showed that -O and -I

mapped

intothe

Us

andmust have beenlocated ina

portion

of the genome that

overlapped

with

BamHI-A, BglI-C,

and SmaI-B. Nevertheless, we detected no

discrepancies

between our data and the map

published by

Fukuchietal. The

resulting partial

EcoRImap is shown below theBamHI,

BglI,

andSmaI maps in

Fig.

4.

EcoRI

fragment

F mapped in the

inverted-repeat

regions

I

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694 SILVA AND WITTER

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:x; :}; ; ::

:: 2:

; f:44:.

i.,; a%,

x.; : :aR>:ss;

.RA.' >..

t, ;' .;..' .-. '.-,;ze

s>C'-;' xe' !--;

..r.;. e;

,;* v}:,-te , s>to

wi} > :'-,: ;w.:..

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Sz .;Z ,;f ,'s@Z:

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*1k.Z.S5;v5>:S

.: :-: .-.{'.'. 's.e -.- .. '- ;', .'-,'.S':;i" ,',

H

-@

B

FIG. 7. EcoRI-Fprobe ofBamHI-digestedDNAfrom oncogenic and attenuated Mdll. 32P-labeled EcoRIfragment F was used to probe BamHI-digestedDNAfromMdll/10-andMdll/81-infectedDEF, asdescribed in the legend to Fig. 5. A,Mdll/10DNA; B,Mdll/81DNA.

(TRL and IRL [Fig. 4]), flanking the long unique region.

Therefore, EcoRI-F should have been present in greater thanmolar quantities. This appeared to be confirmed by the densitometer tracing of the ethidium bromide-stained frag-mentsfromthe EcoRI cutof Mdll/10 DNA(Fig. 2).

Expansion of EcoRI-F. To ascertain the fate of the F

fragment, weused a

32P-labeled

clone ofEcoRIfragment F to probe RE digests of DNA from Mdll-infected cells. A

densitometer tracing ofEcoRI-cut Mdll/10 DNA, probed

witha

32P-labeled

clonecontainingtheEcoRI-F,isshownin Fig. 5A. The DNA was electrophoresed from left to right.

Thesharp peak representsfragmentF. Asimilar

densitome-tertracing of Mdll/81-cutDNAisshowninFig.SB. A broad smear of high-molecular-weight DNA replaced the single, sharpfragment F band. Therefore, it appeared that a heter-ogeneous expansion of the viral genome in the EcoRI-F

region occurredasthe resultofin viro passage. Based on the range of sizes, as measured by gel electrophoresis, EcoRI fragment Fhadbeen expanded by 0.6 to 5.4 kb. Tofurther localizetheexpandedregion,theEcoRI-Fprobewas

hybrid-ized againstBamHI- andBglI-cutDNA.

Fine-structure mapping of the expanded region. A densi-tometer tracing of BglI-cut Mdll/10 DNA, probed with EcoRI-F, is shown inFig.6A. Asimilarexperimentin which

BglI-cut Mdll/81 DNA was probed, is shown in Fig. 6B. The sharpBglI-F of Mdll/10was absent fromMdll/81 and wasreplaced with ahigher-molecular-weight smearofviral DNA. The small BglI fragment M, between BglI-F and

BgII-I,

appeared unchanged in Mdll/10 and Mdll/81 (data

notshown). Therefore, theexpansion occurred in theDNA

region which is incommon betweenEcoRI-FandBglI-F.

The expansion was also mapped totheBamHI RE

frag-ments. A densitometer tracing of

32P-labeled

EcoRI-F

hy-bridized to BamHI-cutMdll/10 DNA is shown in Fig. 7A.

EcoRI-F hybridized toBamHI-D, -H, and -I. The results of

a similarexperiment with BamHI-cut DNAfrom Mdll/81-infected cells is shown in Fig. 7B. BamHI fragment I was

unchanged in Mdll/81. However, both fragments D and H have been replaced by smears of higher-molecular-weight

DNA. These results mapped the expanded region to the small 2.8-kb region which EcoRI-F has in common with BamHI-D and BamHI-H.

To confirm the location ofthe expanded region, double digests ofvirus DNA were probed withEcoRI-F. A

densi-tometertracing ofaportionof the EcoRI and BamHIdouble digestRE patternprobed with 32P-labeledEcoRI-Fis shown

in Fig. 8. Peak 1 (Fig. 8A)was a 4.1-kbfragmentthat was also present in Mdll/81 (Fig. 8B). Peak 2 was a 2.8-kb fragment whichwas missingfrom Mdll/81, confirming the

prediction made from the earlier single BamHI digest. The

broad peak migrating behind peak 1 appeared to be the

expanded2.8-kb fragment. Nocomparable fragments were visible in theMd1l/10DNA. Theseresults,togetherwith the

fact thatthe expanded region wasnotwithin the 1-kb BglI fragment M, enabled us to map theexpanded

region

to the 1.8-kbBglI-BamHI subfragment ofEcoRI-F. The mapof the

EcoRI-F asit wouldappearwithin theIRLis shown in

Fig.

9.

DISCUSSION

Several recentreports have shown that the serial in vitro passage ofoncogenic MDV can result in specific

genomic

alterations. Ross and co-workers found thata6.8-kb EcoRI

fragment was missingin the attenuated HPRS 16/att virus andprobablyresulted from the insertion of repeat sequences

(12). Japaneseworkers havereportedthatafterserial in vitro passage, avirulent MDV wasgenerated throughDNA rear-rangement of BamHI-D and -H in virulent JM and BC-1 viruses (5, 6, 7; Hirai et al., Abstr., Int. Symp. Marek's

Disease, 1984, p. 14). Fortissue culture-attenuated GAand k.M

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GENOMIC EXPANSION OF MDV 695

EcoRI Bqll

I

I

A

BamNI EcoRl

B ~ ~

FIG. 8. EcoRI-Fprobeofdouble-RE-digestedDNA. 32P-labeled

EcoRI fragment Fwas usedtoprobeEcoRI andBamHl

double-di-gestedDNAfromMd11/10-and Md11/81-infectedDEF. The

result-ing autoradiogram was scanned with a microdensitometer, as

de-scribedinthelegendtoFig.5.A,Md11/10DNA;B,Md11/81DNA.

JM viruses, the disappearance of BamHI-D and -H

frag-ments has been shown to be due to heterogeneous DNA

region (Fukuchi etal., Abstr., Int. Symp. Marek'sDisease,

1984, p. 16).

Thisreport bothconfirms and extends the earlierfindings.

(i) We report that it is possible to use total DNA from

MDV-infected cellsfor RE pattern analysis.Thisisareliable

and simplified procedure that circumvents the difficulty of

obtaining cell-free MDV. (ii) By using a published BamHI

mapandthe EcoRI REpattern, wehavepartially mappeda

series of EcoRI plasmid clones. (iii) By using the mapped

clones and the simplified technique for RE analysis, we

fQund that the genome of Mdli is altered as the virus is

serially passaged in vitro. Previous reports of genomic

alterationswere restrictedtovirulentstrains ofMDV.Based

on the induction of gross Marek's disease lesions and the

relatively poor protection provided by turkey herpesvirus

vaccines, the Mdli virus is classified as a very virulent

MDV(15).Weobserved thatuponrepeatedinvitropassage,

the oncogenic Mdll virus lost an EcoRl-F. The alteration

wasmappedtoa1.8-kb regionin the invertedrepeats of the

virus genome and consisted ofheterogeneous DNA

inser-tions, increasing the DNA fragment length from 0.6 to 5.4

kb. Thefaintbandsseenbetween EcoRI fragmentsCandD,

in high-passage MdliDNA, probably representthe

expan-sion ofthe F fragment. Although this genomic expansion

does not appear to impair the in vitro replicative ability of

the virus, thealteration appeared concurrent with aloss of

oncogenicity.

0 1 2 3 4 5 6 7

[image:6.612.59.297.67.373.2]

Kb

FIG. 9. REmapof EcoRIfragmentF.Thelightlyshadedregion representsthe genome location where theexpansionhas occurred.

Absolute correlations ofchangesin theMDV genome with changes in biological properties of MDV are difficult to demonstrate. Serial passage of MDV in cell culture may result in a sequence of biological changes including in-creased growth rate in vitro, inin-creased in vitro host range, decreased growth rate in vivo, loss of A antigen, loss of

membrane antigen, loss of theabilityto spreadby contact, loss of oncogenicity, loss of the ability to replicate at41°C,

and ultimately, complete loss of protective ability and the ability to replicate in the chicken (1, 11; R. L. Witter, in L. N.Payne, ed., Marek's Disease, in press). Most if not allof thesechanges are noncoordinately expressed. Most likely a series of mutations occurs with thoseconferring a selective growth advantage being most likely to be detected. The number and variety of such mutations have not been docu-mented.

The difficulty of trying to associate genomic structural changes withbiological changes has resulted in some confu-sion about therelationship between A antigen disappearance andthe disappearance of the EcoRI-F. The Aantigengene was recently identified by hybridization selection to be associated with a6.8-kb EcoRI fragmentD(R. J. Isfort, R. A. Vrable, K. Nazerian, and L. F. Velicer, Abstr., Int.

Symp.Marek'sDisease,1984, p.21).The authorssuggested thatthis 6.8-kbfragmentisthe same 6.8-kb EcoRI fragment that Ross (12) reported to have disappeared in attenuated HPRS 16/att. However, careful size measurement of DNA

fragments indicates thattheir EcoRI fragmentDis actually

7.8 kb and maps in the long unique region of the MDV genome(Fig. 4).Therefore, although Aantigenwaspresent at least in small amounts in Md1l/81 and Mdl1/93 but was notdetected in Mdll/103 (16),thereductioninthe levels of A antigen would not be related to alterations of EcoRI

fragment F.

Thefour attenuated viruses used in thisstudyrepresented a spectrum ofdifferent biological properties. Mdll/60 still

produced some microscopic nerve lesions in chickens and

spread readily bycontact. These propertieswerelacking in thehigher-passage viruses, yet allfour viruses werehighly protective against challengewithvirulentMDV(16).Thus,it

is interesting that little ifany change in genomic structure was detected among these viruses. However, additional

studies with a variety of different REs may detect further

genomic changes.

Mostimportantly,the observedassociation ofthe EcoRI-F alterationwith the loss ofoncogenicity isconsistent with

thehypothesis thatDNAsequences inEcoRI-F are respon-sibleforMDV tumorinduction. To confirm thishypothesis,

we are in theprocess ofdetermining whether Mdll

oncoge-nicitywill beabrogated byintroducingavarietyof mutations within the 1.8-kbBglI-BamHI subfragment ofEcoRI-F.

ACKNOWLEDGMENTS

Wethank Abby Schwartz for excellent technical assistanceand L. F. Lee fordonatingDNAisolated fromcell-free MDVvirions. WearegratefultoH.J. Kung forthegift oftheEcoRI clones. VOL.54,1985

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[image:6.612.319.554.68.135.2]
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LITERATURE CITED

1. Calnek, B. W., and R. L. Witter. 1984. Marek's disease, p. 325-360.In M.S. Hofstad, H. J. Barnes, B. W.Calnek,W. M. Reid, and tI. W. Yoder, Jr. (ed.), Diseases of poultry, 8th ed. IowaState UniversityPress, Ames.

2. Churchill, A. E., L. N. Payne, and R. C. Chubb. 1969. Immu-nizationagainst Marek's disease using aliveattenuated virus. Nature(London)221:744-747.

3. Fukuchi, K., M. Sudo, Y. S. Lee, A. Tanaka, and M. Nonoyama. 1984. Structure of Marek's diseasevirus DNA:detailed

restric-tionenzyme map. J. Virol.51:102-109.

4. Gibbs,C.P., K. Nazerian, L. F. Velicer, and H. J. Kung. 1984. Extensivehomology exists betweenMarek diseaseherpesvirus and its vaccine virus, herpesvirus of turkeys. Proc. Natl. Acad. Sci. U.S.A.81:3365-3369.

5. Hirai, K., H. Honma, K. Ikuta, and S. Kato. 1984. Genetic relatedness of virulent and avirulent strainsof Marek's disease virus.Arch. Virol.79:293-298.

6. Hirai, K., K. Ikuta, andS.Kato.1981.Structuralchangesofthe DNA ofMarek's diseasevirusdurirng serialpassageincultured cells Virology 115:385-389.

7. Hirai, K., K. Ikuta, andS.Kato.1981.Restriction endonuclease analysis of the genomes of virulent and avirulent Marek's diseaseviruses. Microbiol. Immunol. 25:671-681.

8. Maniatis, T.,E. F.Fritsch, and J. Sambrook. 1982. Molecular

cloning: alaboratory manual. Cold Spring HarborLaboratory, ColdSpring Harbor,N.Y.

9. Nazerian, K. 1970. Attenuation of Marek's disease virus and study of its properties in two different cell cultures. J. Natl. Cancer Inst. 44:1257-1267.

10. Nazerian, K., andL. F. Lee. 1974. Deoxyribonucleic acid of Marek's disease virus inalymphoblastoid cell line from Marek's diseasetumors.J. Gen. Virol. 25:317-321.

11. Payne,L.N., J. A. Frazier, and P. C.Powell. 1976. Pathogenesis of Marek's disease. Int. Rev. Exper. Bio. 16:59-154.

12. Ross, L. J. N., B. Milne, and P. Biggs. 1983. Restriction endonuclease analysis of Marek's disease virus DNA and homology betweenstrains. J. Gen. Virol. 64:2785-2790. 13. Silva,R.F.,and L. F. Lee.1984.Monoclonalantibody-mediated

immunoprecipitation ofproteins from cells infected with Marek's disease virusorturkeyherpesvirus.Virology136:307-320. 14. Witter, R. L. 1982. Protection by attenuated and polyvalent

vaccines againsthighly virulent strains of Marek's disease virus. Avian Pathol. 11:49-62.

15. Witter, R. L. 1983. Characteristics of Marek's disease viruses isolated from vaccinated commercial chicken flocks:association of viral pathotype with lymphoma frequency. Avian Dis. 27:113-132.

16. Witter,R.L.,and L. F. Lee. 1984.Polyvalent Marek's disease vaccines: safety,efficacy and protective synergism in chickens withmaternal antibodies. Avian Pathol. 13:75-92.

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Figure

FIG.1.celldigestedwithDNAMDV RE patterns of purified MDV DNA and MDV-infected DNA. Total DNA from DEF or purified virus DNA was with EcoRI, separated in a 0.6% agarose gel, and stained ethidium bromide
FIG. 2.tractedinphotograph, a EcoRI RE digestion of Md11/10 DNA. DNA was ex- from Mdll/10-infected DEF, digested with EcoRI, separated 0.6% agarose gel, and stained with ethidium bromide
FIG. 4.patternsSmaI-digested Partial EcoRI map of MDV clones. DNA from the pBR328 clones of GA virus was used to probe BamHI-, BgIl-, and DNA from MDV-infected DEF, as described in the text
FIG. 6.andtometer,probeMdll/81DEF. EcoRI-F probe of BglI-digested DNA from oncogenic attenuated Mdll
+3

References

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