Genetic variability of herpes simplex virus: development of a pathogenic variant during passaging of a nonpathogenic herpes simplex virus type 1 virus strain in mouse brain.

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Vol.46,No. 1 JOURNALOFVIROLOGY,Apr. 1983,p.83-93

0022-538X/83/040083-11$02.00/0

CopyrightC1983,AmericanSociety for Microbiology

Genetic

Variability of Herpes Simplex Virus: Development of

a

Pathogenic

Variant During Passaging of a Nonpathogenic

Herpes

Simplex Virus Type 1 Virus Strain in Mouse Brain

H.C. KAERNER,* C. H.SCHRODER,A. OTT-HARTMANN, G. KUMEL,AND H. KIRCHNER

Institute ofVirusResearch,German Cancer Research Center, 6900 Heidelberg,Federal Republic of

Germany

Received 22 July1982/Accepted2December 1982

Herpes simplex virus type 1 ANG(HSV-1 ANG) is originally nonpathogenic for inbred mice upon intraperitoneal intravenous, or intravaginal inoculation. In contrast, micediedof encephalitiswithin 4 to 5 daysafter intracerebral

inocula-tionwiththis strain. HSV-1 ANG wasserially passaged in mousebrains. In two independent series, peripherally pathogenic virus variants had developed and accumulated in the virus progeny after 12 to 15 intracerebral passages. In mixed

infections both nonpathogenic and pathogenic viruses replicated at the primary

site of infection and spread to various organs. However, only the pathogenic

phenotype could be recovered from the spinal cord and the brain. Comparison of therestriction enzymecleavagepatterns of pathogenic ANG andnonpathogenic

ANG virus DNAs revealeddistinctalterations in the S-segment(Us) sequences

bounded by coordinates 0.953 and 0.958 in the prototype orientation and by

coordinates0.862to0.867 in theIs orientationofthe viral genome.However,itis

notknown whether these alterations arephysiologicallyrelevant to the observed

changes in pathogenicity. When coinjected intraperitoneally at 50 to 100-fold

excess, thenonpathogenicHSV-1ANGprotectedmiceagainstitsown pathogen-ic variant as well as against otherpathogenic HSV-1 strains. PathogenicHSV-1 ANG proved to be genetically and phenotypically stable for at least 25 serial passagesin tissue culture at either high or low multiplicityof infection.

It is known from the workofLopez (15-17) and otherinvestigators (11,12) thatanumberof

inbred mice strains differ with respectto their

susceptibilitytoindividual strains ofherpes

sim-plex virus (HSV) upon intraperitoneal (i.p.) in-fection. Conversely, different virus strains

ex-hibit variable pathogenicity in the same inbred

mousehost. Forexample, HSV type1(HSV-1)

WALishighlypathogenicfor6-week-oldDBA/2

mice wheninjected i.p., whereas HSV-1 ANG does not kill the animals under these circum-stances,even atveryhighdosesofvirus(above 107PFUpermouse) (11, 12, 25).The latterfact

has been documented already for very early passages of this strain (passage 3; G. H. von

Mittelstaedt,Ph.D.thesis, Universityof

Heidel-berg,Heidelberg, WestGermany,1972). On the

other hand, intracerebral (i.c.) infection with eitherofthesetwovirus strainsresultsin lethal

encephalitis of mice(7, 23). Therefore, in

addi-tion to thegenetically determinedhost defense mechanisms involved in resistance of mice

againstHSVinfections,virus-encodedfunctions

appear to play a role in different patterns of pathogenicity.

Inthe present paper wedescribethe

develop-mentandproperties ofahighlypathogenic

vari-ant ofa plaque-purified clone of the originally

non-pathogenic HSV-1 strain ANG standard

(21). This variant(HSV-1 ANGpath)originated

in the courseofserial passagesofHSV-1 ANG standard in mouse brain and, in contrast toits parent, kills adultDBA/2miceeven atvery low doses of virus upon i.p. infection. Restriction enzyme analyses revealed that HSV-1 ANG path shows distinct alterations in certainregions

of its genomecomparedwith theparental

apath-ogenicstrain. It alsowill be demonstrated that

both HSV-1 ANG standard and its pathogenic

derivativereplicate similarlyattheprimarysite of infection for some time. However, whereas infectious HSV-1 ANGpathparticlesappear in

thebrain, thenonpathogenicvirus couldnotbe

recovered from thattissue,and thei.p.infected

animals survive.

An additional pathogenic variant of HSV-1

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84 KAERNER ET AL.

ANG was isolated from a second independent

series of viruspassages inmousebrainandwas

found by restriction enzymes analyses to be

indistinguishable from the first ANG path iso-late.

MATERIALSAND METHODS

Virus and cells.Plaque-purified HSV-1strains ANG

standard (21) and WAL (20) were propagated on Africangreenmonkey kidney cells, RC 37 Rita (Ital-diagnostics, Rome, Italy),asdescribed earlier(21).

Mice.DBA/2Jmicewereobtained from G.

Bomholt-gard (Rye, Denmark). Males of 4to 6 weeks ofage

wereusedthroughoutthis study.

Extraction andpurificationofHSV DNA. Extraction

and purification of HSV DNA were performed as describedearlier(21).

Restrictionendonucleases. BamHIwas aproductof

BethesdaResearchLaboratories, Inc.,Rockville,Md.

All other restriction enzymes were purchased from

NewEngland Biolabs Inc., Beverly,Mass.Digestions wereperformed by followingthemanufacturers

proto-cols.

Gel electrophoreses of HSV DNA restriction

frag-ments.Electrophoreseswerecarriedoutin0.5to0.8% verticalagarose(Seakem) slab gelsat40 Vfor 18 hat

room temperature. The gels were stained with

ethi-diumbromide andphotographed under UVlight.

Extraction ofDNA restrictionfragments from agar-osegels.Extraction of DNA restrictionfragmentswas

performed according to Langridge et al. (13). The fragmentswereseparatedinlow-melting pointagarose

gels (BethesdaResearchLaboratories).

Passaging of HSV-1 ANG inmouse brain. Plaque-purifiedHSV-1 ANG standard wasused. About3 x 102PFUin0.05mlof 0.15MNaClwereinoculatedi.c. peranimal. Tenmiceper passagewereinfected. The mice died after1week withinanintervalof2to3days.

The brains of the dead animals were recovered and

homogenizedin a10-fold volumeofsaline. The sus-pensionwas centrifuged at 10,000 x g for10 minat

4°C. The supernatant, which was designated as the

firsti.c.passage,wasused forthe similarpreparation

of subsequent i.c. passages. The virus titers, which weredeterminedonRC 37 Rita cells(19),wereabout5

x 105 to 1 x 106 PFU per brain in all of the i.c.

passages. Two independent series of i.c. passages

wereperformed.

Individual i.c.passages weretestedfor

pathogenici-tyini.p. infectionsofDBA/2mice. About2x103PFU wereinjectedperanimal.Thebrains of micethat had

died within 6 and 10 days after i.p. infection were

recovered andassayedfor thepresence ofinfectious

virus.

Determination of virus titers in differentorgansof i.p.

HSV-infected mice. The progress of i.p. HSV

infec-tions in micewasfollowedby titrating infectious virus particles in the peritoneum, the spleen, the spinal cord, andthe brain. The organswereresected atthe

indicatedtimespostinfection, homogenized, and sus-pended in saline. The suspensions were frozen (-70°C)andthawed threetimes, ultrasonicated for5s (position 4, Branson Sonifier), and centrifuged at

10,000x gfor10min.at4°C.From theperitoneumthe viruswasextensivelywashedoutwith1 mlof saline.

All virustitersweredeterminedonRC37cells(19).

RESULTS

Isolation of an i.p. pathogenic HSV-1 ANG

variant from i.c. virus passages in mice. Two

independent series of i.c. virus passages in DBA/2 mice were performed as described above, starting withplague-purified, peripheral-ly (i.p.) apathogenic HSV-1 ANG standard. In-dividual i.c.passages wereassayed for

peripher-alpathogenicityby i.p. infectionof mice. Inone

of these seriesperipheral pathogenicitywasfirst detectedin the 11th i.c.passage,which killed 30

to 40% of the mice upon i.p. infection with approximately 103 PFUper mouse.

Allearlier i.c.passagesprovedtobe peripher-ally (i.p.) nonpathogenic. In the following i.c.

passages pathogenicity increased rapidly: the

15th i.c. passage and all subsequent serial i.c.

passageskilled100% ofi.p.infected mice.Inthe

second series of i.c.passages, peripheral patho-genicitywas first observed in the 10th i.c.

pas-sage, and afteri.p. infection 100% mortality of

micewas achieved with the 13th i.c. passage.

Virusconstituting the 15th i.c. passageof the first series was plaque purified onRC 37 cells. Of 15 picked clones, 3 proved to be highly

pathogenic in that they killed 100% of mice between 6 and 8dayspost-i.p.infection with 300 PFUperanimal. Theremaining cloneswerei.p. nonpathogenic, like their parental clone HSV-1 ANG standard. Thisfinding suggested that the

15th i.c. passage of HSV-1 ANG in mice still

contained a considerable fraction of i.p.

non-pathogenic virus. Analogous results were ob-tained with the 13th i.c. passage of the second series. The above suggestionwas supported by

restriction enzyme analyses of the viral DNA isolated from the progeny of the 15th i.c.

pas-sage,which will bepresented below.Asjudged

by physical markers, this DNAis amixture of

HSV-1 ANG standard andANGpathgenomes.

From these resultsit becomesmoreevident that both thepathogenic and the nonpathogenic

vari-ants replicate when they are simultaneously

inoculated intomousebrain.

Selective appearance of HSV-1 ANG path in

mousebrainafterperipheral (i.p.)infection with

i.c. virus passages. Groups of 10 DBA/2 mice

were inoculated i.p. with (i) HSV-1 ANG

stan-dard, (ii) HSV-1 ANG path (i.e., one of the pathogenic clones isolated from the 15th i.c.

passage as described above), (iii) the 15th i.c.

ANG passage, and(iv) a1:1 mixture of HSV-1

ANG standard and HSV-1 WAL, as acontrol. Within6 to8days

100lo

of the animals died after infection with thepathogenic ANGvariant,the

15th i.c. passage, and theANG standard-WAL

mixture.

Thebrains ofthemice killedby virus infection

andofthesurvivingmiceinoculated withHSV-1

ANG standard were resected and assayed for

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GENETIC VARIABILITY OF HSV 85

TABLE 1. Peripheral (i.p.) infection of mice withdifferentHSV-1 stocks

Infectious Genotype'/

Infectingviu PFU/ Mice killed virus per peoye fic

mouse per group mouse brain henotypeo

(PFU) Virus

HSV-1 ANG standard 300 0/10 <2

15thi.c. passage 300 10/10 1.2 x 104 HSV-1 ANGpath

HSV-1 ANG path 300 10/10 1 x 104 HSV-1 ANGpath

1:1 mixture, ANG 105each 10/10 1.4 x 104 HSV-1 WAL

standard/WAL

a Asjudged byrestriction enzyme analyses of viral DNAs (see legends, Fig. 2, 3, and 4).

bHSV-1 ANG standard and ANG pathareSyn; strain WAL is Syn+.

the presenceof infectious virus and its respec-tive phenotypes and genotypes. HSV-1 ANG standard and ANGpatharephenotypically Syn, whereas strainWALisSyn+.Aswillbe shown

below, ANG path DNA could be distinguished from ANG standard DNA by restrictionenzyme analyses. The data obtainedaregiven in Table1. The results showthat, after mixed i.p. inocula-tions with either the "homologous" variants HSV-1 ANG standard and ANG path (repre-sented by the 15th i.c. passage)orthe heterolo-gousstrains ANG andWAL, only the pathogen-ic virustypes ANG pathorWAL,respectively, appeared in the brain of mice. Identical results wereobtained with mixtures of ANG standard and ANGpath for i.p. infection.

Thefollowing experimentswerecarriedoutto test whether the inability to recover the non-pathogenic ANG standard virus from mouse

brains reflected the lack ofvirus replication at

the site of inoculationorthe lack of virus spread

toother tissuesorboth.Groups of 10 micewere inoculated i.p. with 103 PFU of HSV-1 ANG

standard and ANGpath, respectively.At

appro-priate times postinoculation, various organs of

the mice were resected and assayed for the presence of infectious virus. The virus titers

found are listed inTable 2. The data suggested

that both HSV-1 ANG standard and its patho-genic variant replicated similarly atthe site of primary infection and mainlyinthespleen of the

animals.Inagreementwith theprevious results,

only the ANG path virus could be recovered

from the mousebrains.

Finally, HSV-1 ANG pathwaspurified by i.p. infection of mice with i.p. pathogenic i.c. ANG standardpassagesandby isolatingthe pathogen-ic virus from the brain of the animals. All 15

clones pickedfrom intracerebral viruswere

like-wise i.p. pathogenic.

A similar selection ofapathogenic variantof HSV-1 ANGwasachievedusingthe 13th of i.c. passages ofthe second series.

Thecloned HSV-1ANGpathwasalsoplaque purified by transfection of BSC-1 cells with

purified viral DNA. The resulting virus stock

proved to be pathogenic, suggesting that the

acquired pathogenicitywasencodedbytheviral

genome.

The i.p. 50% lethal dose in 4- to6-week-old

DBAI2 mice oftheclonedHSV-1 ANGpathwas determinedtobe about 30 PFUpermouse.

Protection of miceagainst lethali.p. infection

withHSV-1pathby coinfectionorpriorinfection

with HSV-1 ANG. As pointed out above, the

15th i.c. passage of HSV-1 ANG (first series), which killed 100% of i.p. infected mice, con-tained about 80%o(12 of 15 subclones) i.p. non-pathogenicvirus. Precedingserial i.c.passages,

being only partially i.p. pathogenic, proved to

containdecreasing fractions of ANGpath.

From this fact itwas suspectedthat the

non-TABLE 2. Spreadofi.p.infection of mice with HSV-1 ANG standard and ANGpath

Titersof infectious virus(PFU/mouse)'

Organ Timei.p. (days) HSV-1 ANG HSV-1 ANGpath

standard

Peritoneal exudate 1 3 x 103 5 x 103

Spleen 1-4 5 x 103 (max) 1 x 104(max)

Liver 1-7 <2 <2

Kidney 1-7 <2 <2

Spinal cord 6 <2 5 x 104

Brain 6 <2 1-3 x 104

a Titersweredeterminedas

described

in thetextand represent averagevaluesof10mice in each case. VOL.46,1983

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TABLE 3. Simultaneous i.p. infection with HSV-1 ANG standard and ANG path at different ratios

Infecting virus Total PFU/_mouse Mice killed_{per group}

HSV-1 ANG stan- 104

dard

ANGstandard/ANG path mixture

1/1 2 x 104 10/10

2/1 3 x104 10/10

5/1 6 x 104 7/10

10/1 1.1 x 105 2/10

50/1 5.1 x 105 0/10

100/1 10.1 X 105 0/10

HSV-1 ANG path 104 10/10

pathogenic virus type could interfere with the

fatal infection ofits pathogenic variant. It has

been shown earlier(20) thati.p. infection with

an approximately50- to 100-fold excess of the

nonpathogenic strain ANG standard protected

mice against simultaneous lethal i.p. infection

with the heterologous pathogenic strain WAL.

The mechanisms involved in this phenomenon

are not understoodatthemoment.

Inanalogous experiments micewere injected

i.p. with different mixtures of HSV-1 ANG standard and ANG path. The results are

com-piled in Table 3 andsuggestthat ANG standard protected theanimals when injected inat least

10- to 50-fold excess over the pathogenic

vari-Hind III

Eco RI

KpnI

BamHi

ant. Regarding the i.p. inoculation with i.c.

passages that resulted in 100% mortality, this

findingsuggeststhatthefraction of ANGpathin these virus passages exceeded 10%of the total infectious virus.

Kinetics of accumulation of HSV-1 ANG path

inthe progenyduring serial i.c. viral passages of

ANG standard: doesANGpath originate

sponta-neously from ANG standard or is it originally

present in the parental clone? There are two

possible explanations for the accumulation of ANG path inmousebrain. Either ANG pathwas

present at a low copy number in the original

ANG standard, due to its spontaneous

genera-tionduringplaquecloning in tissue culture (i.e.,

representingaminor fraction of theprogenyina

single plaque), oritdevelopedduringi.c. repli-cation of ANG standard.

One argument supporting the latter

assump-tion is the fact that a considerable number of individual HSV-1 ANG subclones isolated in

this laboratory in the course of several years

from infected cells proved to be peripherally

nonpathogenic for mice. Further evidence to

this point could be provided by the following

experiments. Five mice were i.c. coinfected

with1 PFUof ANG path and200 PFU ofANG

standard per mouse. As expected, all of the animals diedwithin 3to 4days. Thebrains ofthe

deadanimalswererecovered andmixed

togeth-er.Thetiter ofprogenyviruswasdeterminedto

beabout 1.5 x 105PFUper mouse.The homog-enized brains were used for subsequent serial

H 0-I J A k L D M N, G

J D G ,N F MPLi A I E Hk ,k

G Y E H

ElI$SSNIP

V B L C A ,,T,OI J Ql I F kI

Al Z

E C IIA IMITI 1P,VI1 G IRII DI H __I F WilB ;k'SN, I

D W IY

L

0 0,1 0,3 Q4

Map Units L-S Joint

FIG. 1. Scale maps ofHindIll, EcoRI, KpnI, and BamHI endonuclease cleavage sites on the prototype isomerofHSV-1 ANGDNA.Thejointof theL-andS-segmentsis markedbyaverticalline.

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i.c. passages, injecting 200 PFU per mouse in all cases.The individual i.c. passages were assayed forperipheral pathogenicity by i.p. infection of mice with 3 x 102PFU per mouse. In each of two completely independent series of virus pas-sages thethird i.c. passage displayed about 50% mortality in groups of 10 mice, and the fourth

i.c. passages killed 100% of the animals (i.e.,

contained about 10 to 20% pathogenic virus).

BamH 1 1.8/ AG AROSE

-p.o

.... a

-Ts

--

-_S

-~u

-W.X._{w. .Z}

zB

A

-Y

-A -B

When compared with the results of serial i.c. passaging of the pure ANG standard described above, thesefindingssuggested that (i) the vari-ant clone ANG path is not contained in the

parentalvirus population ofHSV-1 ANG stan-dard but originated spontaneously during i.c.

replication; and (ii) ANG path has a consider-able growth advantage over ANG standard n

mouse brain tissue. Furthermore, it appears

BamH 0,.6/ AGAROSE

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... ...

K

-N.

-N

--a

_---A

-B

-. DC

-F

H G

-Q.,P. __

v--Y

I'

ci

Qi

nL

ci

Ci,

a-FIG. 2. Ethidium bromide-stainedBamHI restriction fragmentpatternsof HSV-1 ANG standard DNA and

HSV-1 ANG pathDNA. Toachieve betterresolution, the fragments were run through 1.8 and0.6%agarosegels.

The nomenclature of the fragments refers to the physical map of BamHI cleavage sites in Fig. 1. Subscript

numbers indicate steps of "band ladders" originating by stepwise amplification of500-bpsequences inthe

inverted S-repeats TRS andIRS(10). The fragmentsconcerned(Fig.1)intheseamplificationsareBamHI-X, -Q, -K and-N (Fig. 1).Arrows markalterations of fragment mobilities in ANG path DNA compared with ANG

standardDNA. VOL.46,1983

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KAERNER ET AL.

likely from the kinetics of the accumulation of

ANG path that its formation is a multiple-step rather than asingle-step event.

Restriction enzyme analyses and mapping of

sequence alterations of HSV-1 ANG path DNA.

Figure1showsvariousrestriction endonuclease

cleavage maps of HSV-1 ANG standard DNA.

Aswas reported earlier (8, 9),theS-segment of

the HSV-1 ANG genome displaysadiscretesize

heterogeneity due to multipleamplification of a

500-base pair (bp)sequencemappingnearthe S-terminus in the S repeatTRs and at the

corre-sponding regioninIRS.Asa consequenceofthis

amplification allDNArestriction fragments

con-E, ..

..-_ %!..i

t::

...Lt

C;g8,~

_. _

FIG. 3. Ethidiumbromide-stainedKpnIandHindIII restriction fragments of HSV-1 ANG standard and ANG path DNAs electrophoretically separated in 0.8 and0.6%agarose gels, respectively. Arrows indicate alterations offragments in ANG pathDNA.As a consequence of theamplification of500-bpsequences inTRs andIRS,the following fragmentsarestepladders of minor bands:KpnI-K, -I,-C, -D; HindIII-B, -C, -E, -F, -G, and -M. In the

casesofhigh-molecular-weight fragments, the ladders are not resolved in these gels and appear as broad smears.

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-1-4w,

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VOL.46,1983

taining this sequence, i.e., S-terminal and L-S

joint fragments, appear as "stepladders" of

DNAbands differing in size by500bp.Recently, A. Podbielski (Ph.D. thesis, University of

Hei-delberg, Heidelberg, West Germany, 1982) in ourlaboratory hasfound that anothersequence

of similar size is amplified in the

TRs-Us

and

IRS-Us boundaries of HSV-1 ANG DNA. All

restriction fragments containingthese

amplifica-tions also form stepladders in agarose gels.

Individualfragments that display this

phenome-non are listed in thelegendsof Fig.2, 3, and 4.

The BamHI, KpnI, HindIII, and EcoRI

re-striction patterns of ANG path DNA are

com-pared with the corresponding ANG standard DNA patterns in Fig. 2, 3, and 4. The

corre-sponding patterns of the ANG path variant

iso-lated during the second series of i.c. passages are identical to those in Fig. 2 to 4 (data not shown). The identity of the altered ANG path

DNA fragments was further verified by blot

hybridizationof KpnIfragmentsFandKand the

totalof BamHIfragmentsof HSV-1 ANG

stan-dard DNA to BamHI restriction patterns of ANG standard andpath(Fig. 5). Thefollowing

alterations wereobserved. (i) ANG path DNA

displays no amplification of the 500-bp

se-quences at both ends of

TRs

and

IRS;

i.e.,

BamHIfragments Q, X, N,and K, EcoRI

frag-ments K, B,C,andH,HindIIIfragmentsB,C,

E, F,G, and M, andKpnIfragmentsK, I,C,and

D are nolonger stepladdersof DNA bands but

form singlebands in therespective gels (Fig. 2,

3,and4). (ii)Thereisadeletion of about 500bp

in ANGpath DNAmappinginBamHI-X(Fig.2

and 5). This deletionapparently does not con-cern EcoRI-K, KpnI-C and -I, BamHI-N, and

HindIII-M, but it does concern HindIII-G and

KpnI-D and -K. It therefore must map exclu-sively in

Us,

spanningthe left end ofBamHI-X,

and doesnotcompriseTRSorIRSsequences.(iii)

Another deletionofapproximately150bp

appar-entlymapsin BamHI-ZofANGpathDNA(Fig.

2 and 5). (iv) Fragment KpnI-F of ANG path DNA is about 230 bp smaller than the

corre-sponding ANG DNA fragment (Fig. 3 and 5). Since KpnI-F overlaps BamHI-Z completely,

one might assume that the 150-bp deletion in

BamHI-Zispartof the230-bpdeletionmapping

inKpnI-F.Theremaining80bpdeleted in

KpnI-F could not be unequivocally localized. Since

neither BamHI-JnorBamHI-N displays altered mobilities, it could concern the small BamHI

fragment of200bp recentlydetectedbyWatson

andVande Woude(26)tomap between

BamHI-Xand -Zandwhichwecouldnotidentifyin our

gels.

Of interest was how far the physical

alter-ations found tobeassociated withthe DNA of

ANGpathcorrelates specificallywiththe

prop-GENETIC VARIABILITY OF HSV 89

erty of peripheral pathogenicity. As shown

above, the 15th i.c. passages ofHSV-1 ANG

proved to be a mixture of about 80o ANG

standard and 20% ANG path phenotypes. In

Fig.6 the KpnI restrictionpatternof the parental

ANG standardDNA is compared with those of

ANG path DNA and the DNA isolated from

virions of the 15thANG standard i.c. passage.

-A.B

C.DE..F.G

H.

_~

I

K,L--.K..K

----K_K

;j

... L

K.

---M

--N

-Oo

cn

::s CL

Q6)

FIG. 4. EcoRI restriction fragment patterns of HSV-1ANG standard and path DNAs in0.8%agarose

gels. Arrows mark fragments altered in ANG path

DNAcompared with ANG standard DNA.

i'liv..110

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90

The latter pattern shows that the total popula-tion of DNA molecules has lost the 500-bp amplifications at both ends of

TRs

and

IRS

found in the parental ANG standard DNA. Fur-thermore, 15thi.c. passage DNA appears homo-geneous with respect to the 500-bp deletion located in KpnI-K. The only detectable evidence that this DNA would represent a mixture of different genotypescomesfromfragment KpnI-F, a minor fraction of which displays a 230-bp deletion similar to that found in ANG path

DNA. As estimated from the intensities of the

deleted and the nondeleted KpnI-F bands in Fig. 6, the relative amounts of the two genotypes

N_* |

K1-n

N- W

''X

z o..$;.... AW.I<<f.:

."

-.. xl

a b c

correlate approximately to the amounts of the corresponding phenotypes ANG path and ANG standard present in the mixture.

The results indicate that the amplification of the500-bp sequenceslacking in

TRs

and

IRS

of theparental ANG genome as well as the 500-bp deletion in KpnI-K are notspecifically associat-ed with the observed peripheral pathogenicity. The only physical marker which can be

tenta-tively correlated to the pathogenic phenotype

from the present results is the 230-bp deletion mapping in KpnI fragment F (Fig. 3 and 5). This assumptionwasfurther supported by restriction analyses of viral DNA extracted from a number

-

K1

0

-Y

-Xi

d e f

FIG. 5. Blot hybridization (23) of32P-labeledKpnI restrictionfragmentsof HSV-1 ANGstandard DNAto

unlabeledBamHI restrictionpatternsof ANG standardandpath DNA, monitored byautoradiography. KpnI

fragmentsFand K(Fig.1)wereisolated fromlow-melting-pointagarosegels (13)andwere32P-labeledbynick

translation (18). Lanesaand c, BlothybridizationofKpnI-Fand-K,respectively,to BamHIrestrictionpatterns

ofANG standard DNA. Lanes d andf,BlothybridizationofKpnI-Fand-K,respectively,toBamHI restriction

patternsof ANG path DNA. Thecomplete BamHI restrictionpatternsof ANG standard andpath DNAs(lanesb

and e, respectively) are visualized by blot hybridization of the total of KpnI restriction fragments of the respectiveDNAs.

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C-del. F L

K L _

-A

t .>

~~~B.C

D.

7=

-Q-I _-E ;_] i~~

-T. U

v

-W.x

-Y.z

- A.

i B',CI

C-'

I.

-r.l

FIG. 6. Comparison ofKpnI restriction fragment

patternsofANGpathDNA and DNA extractedfrom virionsofthe 15th of serialpassagesof HSV-1 ANG

path. Arrowsmark ANGpathDNAfragmentsaltered ascompared with the corresponding ANG standard

DNAfragments.

ofplaque-purified nonpathogenicclones isolated

from the 15th ANG i.c.passage. Figure7shows

the KpnI restriction patterns offive

nonpatho-genicclones. None ofthemdisplaysthe 230-bp

deletion in KpnI-F. However, there is no

evi-denceatthemomentthatthe deletion inKpnI-F

wouldinanyrespectphysiologicallycorrelateto

the alteredpathogenicitypattern of HSV-1 ANG

path.

It should be pointed out that HSV-1 ANG path was found to begenetically and

phenotypi-cally stableduringatleast 25 passages in RC 37 Rita cells in tissue culture performed so far at eitherhigh (10 PFU per cell) or low(0.01 PFU

percell)multiplicity.

DISCUSSION

Infection i.p. of inbred mice with HSV is widely used as amodel to study antiviral defense mechanisms (11, 15, 24, 28). The mice die from encephalitis about 5 to 10 days after i.p. infec-tion with HSV-1. It is well established that there

isconsiderablevariability regarding the i.p.

sus-ceptibility of different mouse strains to

individ-ual HSV-1 strains and vice versa. Similar obser-vations have been reported for other routes of infection byseveral authors (4, 6). In addition to the HSV-1 strain HFEM, which was

demon-stratedby Lopez (15) to be completely avirulent

in i.p. infections for aconsiderable number of mouse strains, it was found in our laboratory thatHSV-1 ANG standard is peripherally

non-pathogenic for mice(12, 20). It should be

empha-sized here that HSV-1 ANG standard is also

nonpathogenicfor mice in intravenous and

intra-vaginal infections (Kumel, unpublished data). We have focused on the possible role of viral

geneticsin thephenomenon of different

suscep-F

-

F

a

b

c

d

e

fg

FIG. 7. KpnIrestrictionfragment

patterns

of

indi-vidualnonpathogenicclones,plaquepurifiedfrom the 15th i.c. passageof HSV-1 ANG standard. The

pat-ters are compared with ANG standard DNA and ANG path DNA as controls. The fragments were

electrophoretically separated on a0.8% agarose gel.

Individualtracks show thepattersof: a, ANG

stan-dard;b-f, nonpathogenicclones 1 to5;g,ANGpath.

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KAERNER ET

tibilities ofmicetoperipheral(i.p.) HSV-1 infec-tions. The development ofahighly pathogenic varient, ANG path, from HSV-1 ANGstandard promisedtobeavaluabletool in such studies. It is of interest that HSV-1 ANG standard was originally avirulent for mice (von Mittelstaedt, Ph.D. thesis). Most recently, 2 of 20 tested HSV-1 strains freshly isolated from patients have beendemonstratedinourlaboratorytobe avirulent for mice in i.p. infections (M.

Batta-Buchle and H. C. Kaerner, unpublished data). Furthermore, we were able to develop i.p. pathogenic variants of both of these strains, which surprisingly originated following similar kinetics asANG path in thecourse of 13 to 15 consecutive i.c. virus passages in mice. From these findings it appears that the property of peripheral virulence is genetically acquired de novo in a multiple-step process by unknown mechanisms involved in the interaction ofthe

viruses with braintargetcells.

Nothing is currently known concerning the

HSV-1viral functions involvedinpathogenicity.

Thephysical alterationsfoundonthegenomeof

ANG path partially mapinregions which have been foundtobe "hotspots"ofgenetic

variabil-ity of HSV-1byanumber ofinvestigators (5, 14, 22)and as yetare physiologicallyundefined. It shouldbe mentioned that the amplification ofa 500-bpsequencelackingatthe S-terminus(TRS)

of the ANG standardgenomeobsereved in ANG

path DNA does not mean the total loss of this

sequence: DNAsequencedataobtained recent-ly in our laboratory (C. P. Gray and H. C. Kaerner, manuscript in preparation) revealed

that this sequence specifies part of the mRNA

encoding for the nonstructural virus protein VP175 (3).

That both the avirulent and virulent variants

replicatetoasimilarextent atthe siteofprimary infection and readily spread to several organs

whereas only infectious virions of ANG path appearin the brainofi.p.infectedmice could be explained in several ways. First, it is possible

that the avirulent strain is unable to infect or

replicate in the cells of the nervous system. A

similarexplanationhas beensuggestedfor

Acy-clovir-resistant and thymidine kinase-negative

HSV mutants by Field and co-workers (1, 2). However, HSV-1 ANG standard is thymidine

kinase positive and replicates readily in mouse

brain,if itis directly injected.Anotherpossible explanation is that strain ANG induces an

ex-traordinary early actinghost defense mechanism

upon peripheral infection which prevents virus

replication in the brain and resultant lethal en-cephalitis, ashas been suggestedby Kastrukoff

et al. (10). One argument against the latter

hypothesis is our finding that 4 days after i.p. infectionwith ANG standardnoviralantigensor

J.VIROL.

antibodies could be detected in the brains of the micebeyond theblood-brain barrier, whereas in the case of ANG path infection the brains of the animals contain significantamounts of both viral antigens and antibodies (H. Fischer and H. C. Kaerner,unpublisheddata).

Ithas also been found that ANG standard and ANG path do not differ with respect to the stimulation of NK cells (Kirchner, unpublished data).

Wewould prefer at the moment the hypothe-sis thatHSV-1 ANG standard can neither repli-cate in the cells of the central nervous system nor betransported axonally to the brain or pass theblood-brain barrier.

Experiments are now under way to introduce the property of peripheral pathogenicity by marker transfer techniques from HSV-1 ANG path and other virulent HSV-1 strains into the genome of ANG and otheravirulent strains to identify genomeregions andfunctions involved in thephenomenonof neurovirulence.

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