Herpesvirus saimiri strain variability.

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Vol.43,No. VIROLOGY, July1982, 352-356

0022-538X/82/070352-05$02.00/0

Herpesvirus saimiri Strain

Variability

RONALDC. DESROSIERS*ANDLAWRENCEA. FALK

Harvard MedicalSchool, NewEnglandRegionalPrimate Research Center,Southborough,Massachusetts

01772

Received 18January1982/Accepted12March 1982

Herpesvirus saimiri was isolated from 22squirrelmonkeys by cocultivation of peripheral lymphocytes with permissive owl monkey kidney monolayer cells.

Comparison ofvirion DNA fragments produced from restriction endonuclease digestion was used as a sensitive measure of strain variability. Although all isolates contained similarities and common features, 19 ofthe 22 were readily

distinguished. Threeof theisolates,however,were

indistinguishable

andpossibly

were related

epidemiologically.

Distinct

subtypes

ofH. saimiriwerenotevident

bythesecriteria;Peruvian,

Colombian,

Guyanan,

and Bolivian

squirrel

monkeys

yieldedisolates without characteristic features

peculiar

tothe

geographic

region.

Threeof three colony-born squirrel

monkeys

thatweretested

yielded

a strainof

virusdistinct fromthatobtainedfrom the mother. Inseparate

experiments,

twoof three animals chosen at random yielded a strain of virus different from that

originally obtained 16 and 22 months

previously; only

oneof the three animals examinedyielded the same strain of virus 22 months after the

original

isolation.

The degree ofrestriction endonuclease

fragment

variability

among H. saimiri

strains appearedtobe greater than

previously

observed for otherherpesviruses.

Herpesvirus saimiri naturally infects most

squirrel monkeys (Saimiri sciureus) in which it causes noapparentdisease.H.saimiriinfection of other species of New World primates

fre-quently results in lymphoma or leukemia.

Squirrel monkeys, the natural hosts, are

com-monly foundinthe rainforestsofSouth Ameri-ca, and animals obtained from different South American regions have previously been shown tohavedistinct physicalappearancesand

karyo-types(15).

Previous analyses ofH. saimiri virion DNA

revealedahighdegreeofintramolecular

hetero-geneity in

guanine-plus-cytosine

(G+C)content

(forreview seereference 11). Unsheared DNA

purified from virions iscomposedofabout90%

infectiousDNA thatbands at adensityof1.705

g/cm3 (45%G+C)inCsClandabout 10%

defec-tive,noninfectiousDNA that bands at adensity

of1.730

g/cm3

(71%G+C) in CsCl. The

defec-tive DNA consists of identical tandem repeat

units of 1.3 kilobase pairs (kb). Because of its high G+C content and density in CsCl, repeti-tive DNA is called H-DNA. Infectious virion

DNA that bands at adensity of1.705

g/cm3

is

composed of 115 kb of unique DNA with an

averageG+C content of36%(density in

CsCl,

1.695

g/cm3)

to which H-DNA repeat units are

covalently attached in the same orientation at each end. Since the number ofH-DNA repeat units ateach endvaries,thesizeofinfectiousH. saimiriDNA is somewhat variable;theaverage

size ofintactinfectious DNA is around 160 kb.

Infectiousvirion DNA is called

M-DNA,

andthe central

unique

115 kb is calledL-DNA.

Analysis

ofsize and number of

herpesvirus

DNA

fragments

generated

by

restriction

endo-nuclease

digestion

has been used

by

others to

distinguish

different strains of

herpes

simplex

virus

(5, 18), Epstein-Barr

virus

(2, 12,

22),

cytomegalovirus

(13),

varicella-zostervirus

(20),

and

equine

herpesvirus

(23),

totracethe

spread

of

herpes

simplex

virus

(3, 5, 17),

andto

estab-lish

equine herpesvirus

subtypes

1 and 4

(23).

Theresults haveindicatedthat

epidemiological-ly

unrelated

herpesvirus

isolatescanbe

readily

distinguished by

the number anddistributionof restriction endonuclease

cleavage

sites intheir

DNAs. This report presents some unique

as-pectsofH. saimiri strain

variability.

AllnewH. saimiriisolatesused inthis

study

(numbered

101 to122in Table 1)wereobtained

from

squirrel

monkeys

of the New

England

Regional

Primate Research Center

(NERPRC)

colony.

The

origin

of

imported

squirrel

monkeys

was determined primarily from phenotypic

ap-pearance and

importation

records; the proper

subspecies

assignment

was further verified for

four of theanimals

by

karyotypeanalysis

kindly

provided

by

N. Ma of NERPRC (Table

1).

ColombianandPeruviansquirrel monkeyswere housedin separate group cages containing 15to

30 members with no

intermixing

of

monkeys

from thetwogeographicregions. It is

possible,

352

on November 10, 2019 by guest

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[image:2.504.59.253.81.392.2]

NOTES 353

TABLE 1. H. saimiri isolates used for analysis of

strainvariability

isolate

_isolate

Animal.no.

Animal noa Origin of_animal Comment_referenceor no.

101 458-78 ColonybornOffspring of 385-70

102 197-71 Guyana Karyotyped

103 119-70 Peru Karyotyped

104 385-70 Peru 105 201-71 Guyana

106 254-71 Peru 107 256-71 Peru

108 302-78 Colonyborn Offspring of254-71

109 417-78 Colonyborn Offspring of256-71

110 483-77 Colombia

111 24-69 Colombia Karyotyped

112 505-77 Colombia

113 488-77 Colombia

114 494-77 Colombia

115 634-71 Peru 116 482-77 Colombia

117 484-77 Colombia

118 486-77 Colombia

119 487-77 Colombia

120 29-76 Bolivia 121 24-76 Bolivia 122 487-80 Bolivia

11 ? ? 10

S295C 83-67 ? 19

SMHI 139-67 ? 6

OMI Owlmonkey ? 14

isolate

aThe last twodigits of the animalnumbersrefer to

theyear theanimalwasacquiredbyNERPRCorborn

into the colony. For example,squirrel monkey254-71

wasimportedfrom Peru in1971,andsquirrel monkey 302-78wasbornin 1978.

however, that many of these animals were

ex-posedtosquirrelmonkeysfromother

geograph-ic regions, forexample, during importation or

through contact with adjacent cages. Squirrel

monkeys 458-78, 302-78, and 418-78, born to

Peruvian parents, were kept in the samegroup cagewiththeirPeruvianparentsand other

Peru-vian squirrel monkeys.

Peripheral lymphocytes were separated fiom

whole blood collected from apparently healthy

squirrelmonkeys by bandingover Ficoll-Hypa-que (density, 1.076 g/cm3). Lymphocytes were

cocultivatedwith monolayersofpermissive owl

monkey kidney cells in culture until

virus-in-duced cytopathic effect was complete.

Virus-induced foci of the cytopathic effect usually becameevident 5to10daysafter cocultivation.

More than 90%o ofvirus rescue attempts were successful. The celllysates wereused as virus

stock to inoculate owl monkey kidney cells in

150-cm2 flasks for purification of virions and

extractionof virionDNAaccordingto

previous-lypublished procedures (1).

Virion M-DNA wasanalyzedwith the follow-ing restriction endonucleases:

SmaI,

KpnI,

BamHI,

EcoRI, HpaII,

Sacl,

TaqI,PstI, HpaI,

and FnuDII. Maps have been published for strains 11, S295C, OMI, and SMHI by using

restrictionendonucleases

SmaI,

KpnI,

BamHI,

andEcoRI (11). These last fourenzymes were

sufficientfordistinguishingmany of theisolates,

butthe other enzymes which cleave H. saimiri

DNA more frequently were used to establish more definitively the identity or nonidentity of any two isolates.

Sacl,

HpaII, and EcoRI +

SmaIwerechosen to depict the strainvariability

(Fig. 1). Of 22 isolates, 19 were readily distin-guished by these criteria, and when the 4 previ-ously used laboratory strains were included in the analysis, 23 of the 26 isolates were

distin-guishablefrom one another. Three new isolates (102, 106, and 109) could not be distinguished

and appeared to be identical. Isolate 106 was obtained from an animal born in the Peruvian

group cage. It seems likely that these three animals wereinfected by the same strain of virus throughsomecommonsource atNERPRC,but the

possibility

that this virus strain was intro-duced into the colony independently by more than oneimported animal cannot beexcluded.

Based on themolaritiesoffragmentsand sum molecularweights,thepatterns shown in Fig. 1 and others not shown weregenerallyconsistent

with asingle virus strain beingpresent in each isolation. This is in spite of the facts that viruses were not plaque purified and that a crude cell

lysate

was used forpreparation of viral DNA.

Only isolate 104 gave several less-than-molar

fragments, suggesting that more thanone virus

strain was present (Fig. 1). Also, isolate 113

yielded an unusual Sacl fragment greater than two molar; this probably arose from a

popula-tion ofdefectiveDNAmolecules(Fig. 1).

Sufficient similarity existed among these

iso-lates to certify that we were indeed

analyzing

variousstrains ofH.saimiri. Forexample, SmaI

characteristically

cleaved several times in each repeat unit of H-DNA but did not appear to

cleaveL-DNA; the

supermolar fragments

inthe

lowerportionof the EcoRI + SmaI

gel

ofFig. 1

were derivedfrom the

repetitive

H-DNA.

Nev-ertheless, a single discriminating enzyme such as

HpaII

or Sacl was usually sufficient for

distinguishinganytwononidenticalisolates.

Dif-ferences in the degree ofvariability observed withenzymes thatcleavedwithsimilar

frequen-cywere

consistently

noted. For

example,

most

differences in EcoRI

fragments

among strains

could be

explained by

a

simple gain

orloss of one tothree cleavage sites. WithBamHI,

how-ever,twoisolateswere

frequently

found to differ at

50%o

or more of the

cleavage

sites (datanot

shownandseereference

11).

The

greatest

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NOTES 355

ability among isolates was observed with the

restrictionendonuclease HpaII. As anextreme example, strain S295C yielded 16 HpaII frag-mentsgreater than 1 kb, and strain 11 yielded 17 HpaII fragments greater than 1 kb, with total sizes ofaround 115 kb; yet only twofragments comigrated on agarose gels (Fig. 1). This is

remarkablesince thedigestionpatterns of these twostrains with otherenzymes aresimilar,- and

hybridization analyses have indicated a high degree ofcrosshomology(>90%o)withless than

3% base mismatch(16).

The variability in restriction endonuclease cleavage sites is certainly greater among H. saimiri strains than that previously observed

amongstrains of Herpesvirus tamarinus, a her-pes simplex-related virus also indigenous to

squirrelmonkeys (7). Thevariabilityincleavage patterns also appears to beconsiderablygreater than previously published for herpes simplex

virus (5, 18), Epstein-Barr virus (2, 12, 22),

equine herpesvirus (23), cytomegalovirus (13),

andvaricella-zoster virus (20).

Unlike someotherviruses, herpesvirusesare noteasily spread from individual to individual

throughcoughing, sneezing,etc., and it has been

hypothesizedthat the spread of herpes simplex virus within a region or community may be

restricted, beinglimitedtofamilymembersand

close contacts (21). To obtainevidence on the

frequency ofoffspring infectionfrom the moth-er, H. saimiri isolates obtained during the 1st year of life from three colony-born squirrel monkeys (101, 108, and 109) were compared withisolatesobtained simultaneouslyfromtheir mothers (104, 106, and 107). Offspring were

nursed by their mothers in the same Peruvian

group cage. None of the three

colony-born

squirrel monkeys

yielded

the same strain of virus isolated from the mother. Isolate 109, although different fromisolate 107 obtained from theoffspring's mother, wasthe same as isolate 106obtainedfromanother mother(Table 1 and

Fig. 1). These results may not be

surprising,

since a

considerable

amount of interaction

oc-cursbetween

offspring

and all the other animals in a groupcage. However, wecannot be sure

that these

offspring

were not infected by their

mothers at some

time,

since results

presented

below indicate that

squirrel monkeys

may be

infectedwithmorethanonestrain of H. saimiri

or readily become reinfected with a different strain ofH. saimiri.

It was not

possible

to group the H. saimiri

isolatesinto

subtypes

basedonsimilarities in the

restrictionendonucleasefragmentation

patterns.

Furthermore, no characteristic features were

notedinisolatesobtainedfrom

squirrel monkeys

imported

fromthedifferent

geographic

regions.

Squirrel monkeys

197-71,

488-77, and 484-77

were bled for additional virus isolations at22, 16, and 22months,respectively,after the origi-nal isolations. Isolates obtained from animal 484-77wereidentical at the two different times, butdifferentstrains of virus wereobtainedfrom animals 197-71 and 488-77 at the two bleedings (datanotshown).Thesecond bleedingof197-71

yielded an isolate apparently identicaltoisolate 105fromsquirrelmonkey201-71; although these two animals were imported at the same time, there is norecord of their beinghousedtogether

at NERPRC. The second bleeding of 488-77 yielded an isolate different from any other iso-late wehaveexamined.Thenumber ofcleavage

site differences observed in isolates from ani-mals 197-71 and488-77 atdifferent times is too large to be reasonably accounted for by alter-ations of a virus strain while present in the animal. Marmosets infected with anattenuated

strain of 11 stably retain restriction

endonucle-ase cleavage sites as long as 4 years after the

original infection (9; data not shown). Thus, these results indicated that animals 197-71 and 488-77 had been infected with more than one strain of H. saimiri. We cannotdeterminefrom these data whether each animal was infected with two or more strains of virus before our

originalbleeding, with only one strain

predomi-nating at any one time, or whether the animals were reinfected exogenously after the original bleeding, with the reinfecting virus becoming predominant. Upon examination ofherpes

sim-plex virus type 2 recrudescent lesions, two of

eight patientsyielded different strains ofviruson

successive isolations(4). These results withH.

saimiriand herpes simplexvirus do not

neces-sarily

meanthat the immune system isinefficient

inpreventing exogenous reinfection bya

differ-ent strain of herpesvirus. It is

possible

that

seronegative individualscanbeinitially infected

withseveraldifferent strainsof virus

during

the

susceptible

period,

but

only

oneofthesestrains

may predominate duringavirus isolation. Fur-therwork isneededto

distinguish

these

possibil-ities.

weare gratefulto SharonTracy,Joseph Eannuzzo,and

Daniel P. Silva fortechnicalassistance, Carel Mulder and Nancy Ma for helpful discussion, and Linda Rennie for

preparationof themanuscript.

These studies were supported by Public Health Service

grantRR00168from theDivisionofResearchResourcesto NERPRC, byPublic HealthServicegrant R01CA27225-02 from theNational Institutes ofHealth,byagrantfrom the MiltonFund,andbyafellowshipfrom theMedical

Founda-tion,Inc.,ofBoston.

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