INFECTION AND IMMUNITY, Sept. 1976, p. 759-766Copyright © 1976 American Society for Microbiology

Vol. 14, No. 3Printed in U.S.A.

Isolation and Characterization of Two New Herpes-LikeViruses from Capuchin Monkeys

M. A. LEWIS, L. D. FRYE,1 C. J. GIBBS, JR.,* S. M. CHOU, E. C. CUTCHINS, D. C. GAJDUSEK,AND G. WARD

National Institute ofNeurological and Communicative Disorders and Stroke, National Institutes ofHealth,Bethesda, Maryland 20014*; University of West Virginia, Morgantown, West Virginia 26505;

Catholic University ofAmerica, Washington, D.C. 20017; and Public Health Research Institute of theCity ofNew York, Inc., Otisville, New York 10016

Received for publication 14 April 1976

Two herpes-like viruses were isolated from capuchin monkey (Cebus apella)brain and (Cebus albifrons) spleen cell cultures, respectively. Both isolatesinduced similar cytopathic effects consisting of rounded and ballooned cells inthe original monkey cell cultures and in a wide range of permissive cell types.Neutralizing antibody to each virus was present in serum from the capuchinmonkey from which it was isolated, but the two viruses did not cross-react byneutralization. Fluorescein isothiocyanate conjugates of hyperimmune rabbitserum to one of the isolates showed an antigenic cross relationship between thetwo isolates. By electron microscopy, herpes-like virus particles were observedin the nucleus and cytoplasm of infected human diploid fibroblast cell cultures.Virus-infected cell cultures stained with acridine orange revealed small deoxyri-bonucleic acid-containing intranuclear inclusion bodies. Both viruses were in-hibited by 5-fluorodeoxyuridine and inactivated by chloroform or exposure to56°C for 30 min. Antisera prepared against 16 prototype herpesviruses andcytomegaloviruses did not neutralize approximately 100 50% tissue cultureinfective doses of either capuchin isolate. Neutralizing antibody to the capuchinisolates was detected in sera from 8 of 17 capuchin monkeys but not in sera from16 humans, 15 chimpanzees, and 10 spider, 6 rhesus, and 5 squirrel monkeys.

Reports from other laboratories have de-scribed the isolations of 19 herpesviruses from10 species of old- and new-world monkeys (1, 11,15); several of these viruses induced acute en-cephalitis and neoplastic disease in experimen-tally inoculated animals (19, 20, 28). Nuclearinclusions resembling those produced by cyto-megalovirus have been observed in the salivaryglands of the capuchin monkey Cebus fatuella(8), but until recently no virus of the herpesgroup had been isolated from the capuchinmonkey. In this report we describe the isolationand characterization of two herpesviruses fromcapuchin monkeys. By neutralization proce-dures these viruses were antigenically distinctfrom each other, as well as from other simianand human herpesvirus types tested.

MATERIALS AND METHODSExplant culture preparation. Cortical brain tis-

sue taken at necropsy from capuchin monkey AP-18was minced into small pieces and explanted intoFalcon flasks as previously described (26). Spleentissue taken at autopsy from capuchin monkey AL-5

I Present address: University of Southern CaliforniaSchool of Medicine, Los Angeles, CA 90032.

was fragmented with forceps and explanted into 60-mm Falcon plastic tissue culture dishes containing 3ml ofW/05 medium (7). The cultures were incubatedat 35°C in an atmosphere containing 5% CO2, andfluids were changed twice weekly using Eagle mini-mal essential medium supplemented with penicillin(100 U/ml), streptomycin (100 ,ug/ml), and 20% fetalbovine serum inactivated at 56°C for 60 min. Whenthe cell monolayers were about 75% confluent, thecells were treated with a mixture of 0.05% trypsinand 0.02% ethylenediaminetetraacetic acid and sub-cultured into new flasks (21).

Viruses. Undiluted cells and supernatant fluidsfrom the capuchin monkey cell cultures were inocu-lated into human foreskin (MA-184) cell cultures.MA-184 tissue culture stationary tubes were rou-tinely used for preparation of virus pools and forviral infectivity titrations.

Other viruses used in this study were: herpessimplex virus (Mayo-1814 strain), vesicular stoma-titis virus (Indiana strain), poliovirus type I, vacci-nia virus (Wyeth strain), parainfluenza virus type I(HA-2 strain), measles virus (Ostereich strain), ade-novirus Pan 7 (4), and simian foamy virus type 7(13).

Tissue culture studies and media. The followingcell lines were used in viral susceptibility studies:human skin and muscle (MA-337), human foreskin

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(MA-184), human embryonic lung (WI-38), humanembryonic kidney, primary rabbit kidney, continu-ous African green monkey kidney (Vero), Africangreen monkey fetal lung (MA-117), and horse testes(MA-188) from Microbiological Associates, Inc., Be-thesda, Md. Flow Laboratories, Rockville, Md., sup-plied human fetal brain (Flow 3000), continuous pigkidney (PK-15), and whole mouse embryo. Owlmonkey kidney, squirrel monkey kidney, and squir-rel monkey fetal lung were kindly supplied by LouisMelendez, New England Regional Primate ResearchCenter. Rhesus monkey fetal lung cells were estab-lished in our laboratory. Human embryonic kidneyand primary rabbit kidney were maintained withbasal medium of Eagle supplemented with penicillin(100 U/ml), streptomycin (100 ,ug/ml), and 5% fetalcalf serum inactivated at 56°C for 60 min and werefed twice weekly. All other cell lines were main-tained in Eagle minimal essential medium supple-mented with penicillin (100 U/ml), streptomycin(100 A.g/ml), and 2% inactivated fetal calf serum.For plaquing studies, 35-mm Falcon petri plates

containing confluent WI-38 cells were inoculatedwith 0.5 ml of serial 10-fold dilutions of AP-18 andAL-5 isolates. Plates were incubated at 35°C for 1 h(shaking at 15-min intervals); the inoculum wasremoved and the plates were overlaid with 0.75%Methocel (24). Cell monolayers were fixed, stainedwith Giemsa, and examined for plaques at 5-, 7-, and10-day intervals.

In vivo studies. Litters of 2- to 3-day old, general-purpose NIH Swiss albino mice were inoculated viathe intracerebral (0.02 ml) or intraperitoneal (0.05ml) routes with 104-5 50% tissue culture infectivedoses (TCID50) of the capuchin isolates per ml andobserved for 9 months for overt signs of clinicaldisease. Mice were bled out, and the terminal serawere tested for neutralizing antibody to AP-18 andAL-5 viruses. Three neonatal guinea pigs were in-jected in the footpad with 0.2 ml of undiluted virus,held for 1 year, and autopsied. The scarified corneasof three rabbits were infected with undiluted virus,and the animals were held for 1 year and autop-sied. Terminal sera were tested for neutralizing an-tibody to the AP-18 and AL-5 viruses.

Nine-day-old embryonated hens' eggs were inocu-lated via the allantoic and amniotic cavities, theyolk sac, and the chorioallantoic membrane. Allan-toic and yolk sac fluids and chorioallantoic mem-branes were harvested on days 3, 5, and 7 and inocu-lated into stationary tube cultures of MA-184 cells.Sera from chickens inoculated during gestationwere collected 2 days after hatching and tested forneutralizing antibody to the AP-18 and AL-5 vi-ruses.

Physicochemical characterization. To determinethe size of the viral isolates, supernatant fluids fromAP-18 and AL-5 virus-infected cell cultures werespun at 1,000 rpm and filtered through typeHAWP450, GSWP220, and VCMP100-mm mem-brane filters (Millipore Corp., Bedford, Mass.) (6).For electron microscopy studies, virus-infected andcontrol MA-184 cell cultures were detached into themedium with a rubber policeman. The suspendedcells were pelleted by centrifugation, fixed in 2.5%

glutaraldehyde for 30 min, and suspended in Mil-lonig's buffer (22). The cells were postfixed in 1%buffered osmium tetroxide for 1 h, dehydrated ingraded ethanol, and embedded in an epoxy resin(Epon-Araldite) mixture. Ultrathin sections stainedwith uranyl acetate and lead citrate were examinedwith an electron microscope (AEI-Corinth).

Viral sensitivity to 5-bromo-2-deoxyuridine(BUdR), 5-fluoro-2-deoxyuridine (FUdR), chloro-form, and temperature was determined by methodspreviously described (5, 10, 12, 29). Aminopterin(Lederle Laboratories) was used at a 6 x 105 Mconcentration in W/I5 medium lacking folic acid,hypoxanthine, thymidine, and fetal bovine serum;either BUdR or thymidine, respectively, at a finalconcentration of 6.4 x 10-2 M or 10-5 M, was alsoincorporated into the medium (7). Standard viralhemagglutination and hemadsorption assay tech-niques were used (27, 32). To test for the presence ofinclusion bodies, 75-cm2 Falcon flasks of WI-38 cellsinoculated with either the AP-18 or AL-5 virus werefixed in absolute methanol at different stages ofcytopathic effect (CPE) and were stained by a modi-fication of the acridine orange technique (18).

Serology. For the preparation of hyperimmunesera, rabbits were injected with undiluted virus ininterdigital spaces (1.0 ml) and interscapularly (1.0ml) (9). Starting at day 21, rabbits were givenbooster injections intravenously with 1.0 ml of undi-luted virus at weekly intervals for 5 weeks and wereexsanguinated 2 weeks after the last intravenousinjection. Fluorescein isothiocyanate conjugateswere prepared from the hyperimmune rabbit serumusing standard methods (6a). Fluorescent antibodystudies were conducted according to the method ofTheil and Smith (30).

Standard herpesvirus neutralization tests wereperformed using 100 TCID5/, of the AP-18 and AL-5viruses, respectively, and serial twofold dilutions ofsera inactivated at 56°C for 30 min (31). Serum fromrabbits hyperimmunized with the AP-18 and AL-5viruses was tested for complement-dependent neu-tralizing activity as described by Anderson (2). Thefollowing prototype herpes antisera, which weretested for neutralizing activity against the AP-18and AL-5 viruses, were supplied by Louis Melendez:Herpesvirus tamarinus, H. saimiri types I and II, H.ateles, H. simae, H. aotus types I and II, H. hoministype I, H. canis, H. suis, owl monkey kidney isolate,sand rat nuclear inclusion agent, ground squirrelagent, infectious bovine rhinotracheitis virus, andmicrotus mouse kidney isolate. Serum from arhesus monkey with high-titering neutralizing anti-body against rhesus monkey cytomegalovirus wasprepared in our laboratory (3).

Sera from chimpanzees, spider monkeys, rhesusmonkeys, capuchin monkeys, and squirrel monkeyshoused in our animal facilities and sera from animalcaretakers and laboratory personnel were screenedfor neutralizing antibody to the isolates.

RESULTS

Cytopathogenic changes were observed in thesecond subcultures of AP-18 brain on day 54

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after explanting. AL-5 spleen cultures began toshow CPE in the original petri dishes approxi-mately 1 month after explanting. In both, smallclusters of cells lost their normal fibroblasticmorphology, became stellate, and later becamerounded and detached from the substrate. TheCPEs of the two viruses (AP-18 and AL-5 iso-

lates) were identical in all permissive cell types(Fig. 1).Both isolates induced CPE in human skin

and muscle, fetal brain, lung, embryonic kid-ney, and foreskin, in squirrel, African green,and rhesus monkey fetal lung, and in horsetestes, rabbit kidney, and whole mouse em-

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762 LEWIS ET AL.

bryo. Squirrel and owl monkey kidney, Vero,and PK-15 were not susceptible to the isolates(no recognizable CPE).Both AP-18 and AL-5 viruses produced small,

clear, homogeneous plaques in WI-38 cells over-laid with 0.75% Methocel and fixed and stainedat 5-, 7-, and 10-day intervals. Plaques wereevident 5 days after inoculation, and the AP-18and AL-5 viruses each had titers of 2 x 105plaque-forming units/ml 10 days after inocula-tion.Experimental hosts. Litters of 2- to 3-day-old

mice inoculated intracerebrally or intraperito-neally with the AP-18 and AL-5 isolates failedto develop overt signs of clinical disease whenobserved for over 9 months. Inoculated guineapigs and rabbits developed normally over thecourse of 1 year. Neutralizing antibodies to AP-18 and AL-5 viruses were not observed inmouse, guinea pig, and rabbit sera obtainedfrom inoculated animals.Embryonated hens' eggs inoculated with the

AP-18 and AL-5 viruses by the allantoic, am-niotic, yolk sac, and chorioallantoic membraneroutes developed normally without disease orabnormalities. Attempts to reisolate the virusin MA-184 cells from allantoic and yolk sacfluids and chorioallantoic membranes of inocu-lated eggs were negative. Neutralizing antibod-ies to AP-18 and AL-5 viruses, respectively,were not detected in the serum of chickenshatched from eggs inoculated with homologousvirus during gestation.

Viral characteristics. The biological andphysical properties of AP-18 and AL-5 virusesare summarized in Table 1. As determined byfiltration procedures using Millipore filters,both AP-18 and AL-5 viruses range in size be-tween 100 and 220 nm in diameter. These datawere confirmed by electron microscopy, inwhich enveloped particles 180 nm in diameter,morphologically similar to herpesviruses, wereobserved in the cytoplasm and on cell surfaces.Numerous cores 80 nm in diameter and capsids100 nm in diameter were observed in the nucleiof infected cells. The majority ofthe capsids hadan electron-lucent core, and a few particleswere seen in the perinuclear space where her-pesviruses are usually observed budding intothe cytoplasm (Fig. 2).The AP-18 and AL-5 viruses were resistant to

treatment with BUdR (6.4 x 10-2 M), whereasBUdR-treated herpes simplex virus displayed a1.8 log1, inhibition, which was completely re-versed by the addition of thymidine. The vesic-ular stomatitis virus control was unaffected bytreatment with BUdR.Treatment with FUdR (10-2 M concentra-

tion) resulted in a 1.5 log,(, inhibition ofthe AP-

TABLE 1. Biological properties ofAP-18 and AL-5viruses

Property AP-18 AL-5Size by filtration (nm) <220->100 < 220-> 100Size by electron micros- 180 180copy (nm)

Nucleic acid type DNAa DNA(FUdR sensitivity)

Chloroform sensitivity Labile LabileHemagglutinin Negative Negative56°C/30 min Labile LabileInclusion bodies (acri- Intranuclear Intranuclear

dine orange)Pocks on chorioallantoic Negative Negativemembranea DNA, Deoxyribonucleic acid.

18 isolate, a 3.0 log,,, inhibition of the AL-5isolate, and a 1.3 log10 inhibition with herpessimplex virus. The addition ofthymidine signif-icantly reduced this inhibition. Vesicular sto-matitis virus control was not inhibited byFUdR.

Since it was suspected that the isolateslacked a means of phosphorylating the BUdR,another inhibitor, the folic acid antagonist ami-nopterin, was used to account for AP-18 andAL-5 BUdR resistance. Aminopterin plusBUdR were inhibitory, whereas aminopterinplus thymidine or BUdR alone were not.

Chloroform treatment completely inacti-vated 2.5 log1O of the AP-18 isolate and 4.5 log1Oof the AL-5 isolate. Simian foamy virus type 7was decreased by 3.0 log1o, whereas 5.3 log1o ofthe adenovirus Pan 7 was unaffected. Cell-freepreparations of AP-18 and AL-5 viruses werecompletely inactivated after exposure to 56°Cfor 30 min.AP-18 and AL-5 viruses failed to hemagglu-

tinate guinea pig, chicken, human 0, or rhesusmonkey erythrocytes tested at 0.5 and 1% con-centrations at 4, 22, and 37°C. AP-18 and AL-5virus-infected MA-184 cells did not hemadsorb0.5% suspensions of the same erythrocyteswhen incubated at 4 or 37°C. Parainfluenzavirus type I- and measles virus-infected MA-184cells, used as positive controls, gave expectedresults.

Intranuclear inclusion bodies of the herpesvi-rus type were observed in WI-38-infected cellsafter staining with acridine orange. Their pres-ence was not detectable until approximately50% of the cells showed CPE, and as the CPEprogressed all cells appeared to contain thesedeoxyribonucleic acid bodies (Fig. 3).

Serological properties. Serum from capu-chin AL-5 completely neutralized the AL-5 iso-late at a 1:16 dilution, and serum from capuchinAP-18 completely neutralized the AP-18 isolate

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FIG. 2. Electron micrograph ofAP-18 virus-infected MA-184 cells. The nucleus is filled with capsids 100nm in diameter containing hexagonal-shaped cores that are often electron lucent. In the upper left-handcorner of the x93,600 magnification, two particles are present in the perinuclear space.-

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FIG. 3. Deoxyribonucleic acid-containing intranuclear inclusion bodies in WI-38 cells infected with AP-18 virus at 37°C and stained with acridine orange 5 days after inoculation. x3,700.

at a dilution of 1:10 (Table 2). The AL-5 isolatewas not neutralized by a 1:2 dilution of AP-18serum, and the AP-18 isolate was not neutral-ized by a 1:2 dilution of AL-5 serum. Sera at a

dilution of 1:2 from rabbits hyperimmunizedwith the AP-18 and AL-5 viruses did not neu-

tralize 100 TCID5O, of either virus.Rhesus monkey cytomegalovirus antiserum

and the following prototype herpesvirus anti-sera, screened at dilutions of 1:10, also failed toneutralize 100 TCID5,, units of the AP-18 andAL-5 isolates: H. tamarinus, H. saimiri types Iand II, H. ateles, H. simae, H aotus types I andII, H. hominis type I, H. canis, H. suis, owlmonkey kidney isolate, sand rat nuclear inclu-sion agent, ground squirrel agent, infectiousbovine rhinotracheitis virus, and microtusmouse kidney isolate.

Neutralizing antibody to AP-18 virus was de-tected in the sera of 8 of 17 capuchin monkeystested at a 1:2 dilution. Furthermore, two of thecapuchin monkeys from this group neutralizedthe isolate at a 1:4 dilution, whereas one mon-

key serum neutralized the isolate at a 1:8 dilu-tion. Neutralizing antibody to the AL-5 viruswas detected in only 2 of 16 capuchins tested,and these two animals were among the group of

TABLE 2. Cross neutralization studies ofAP-18 andAL-5 viruses

Serum-neutralizing antibody titeraVirus

AL-5 AP-18

AL-5 1:16 < 1:2AP-18 <1:2 1:10

a Reciprocal of highest dilution of serum thatcompletely neutralized 100 TCID50 units of virus.

nine animals that did not have neutralizingantibody to AP-18 virus; indeed, in no instancedid we detect a single animal whose serumneutralized both viruses. Neutralizing anti-body to AP-18 or AL-5 viruses was not detectedin serum from 7 animal caretakers, 9 otherlaboratory personnel, 15 chimpanzees, 6 rhesusmonkeys, 10 spider monkeys, and 5 squirrelmonkeys tested at 1:2 dilutions.

Fluorescein isothiocyanate conjugates of AP-18 rabbit serum, when used at a 1:20 dilution indirect immunofluorescence, showed an anti-genic cross relationship between the AP-18 andAL-5 isolates. The AL-5 conjugate is in prepa-ration.

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DISCUSSIONThe size and morphology of these isolates,

their sensitivity to temperature, chloroform,and FUdR, and the presence of deoxyribonu-cleic acid-staining nuclear inclusion bodiesidentify them as members of the herpesvirusgroup. The distinction between herpesvirusesand cytomegaloviruses is still an arbitrary oneat best. Several reviewers have attempted todefine the differences between these twosubgroups (14, 23, 33). Plummer et al. (25)found no difference in the deoxyribonucleic aciddensity or behavior in tissue culture of 14 her-pesviruses representing both subgroups. Usingthe criteria of Hsiung et al. (14) for the divisionof the herpesviruses into groups A (herpesvi-ruses) and B (cytomegaloviruses), these iso-lates appear to fit into group A on the basis ofparticle size and into group B on the basis ofabsence ofpocks on chorioallantoic membranes.The distinction is less clear when one considersthe criteria of virus maturation and release. Inearly passages in MA-184 cells, the isolates ma-tured and were released slowly, but by passage6 complete destruction of the MA-184 cell sheetoccurred within 48 h. The characteristic swell-ing of cells and large intranuclear inclusionsassociated with cytomegaloviruses were not ob-served, but, rather, small intranuclear inclu-sions reminiscent of herpes simplex virus werepresent. In view of these differences, the twocapuchin monkey isolates are referred to as"herpes-like" viruses rather than being identi-fied as belonging to either the herpesvirus orcytomegalovirus subgroup.

It is interesting to note that neither isolate issensitive to BUdR but both are inhibited bytreatment with FUdR. When the endogenoussynthesis of deoxythymidine 5'-monophosphateis inhibited by FUdR, the growth of the virusbecomes dependent on externally supplieddeoxythymidine as well as on uridine. Since itis suspected that the AP-18 and AL-5 isolateslack the thymidine kinase gene, they cannotutilize external deoxythymidine and thus areinhibited by medium containing FUdR. To ex-plore this possibility, another inhibitor, thefolic acid antagonist aminopterin, was utilized.Since aminopterin blocks the de novo synthesisof deoxyribonucleotides, this inhibitor "forces"the cell to utilize "scavenger" pathways for thy-midine 5'-triphosphate production by phospho-rylation ofthymidine (16). Without drug inhibi-tion, the BUdR may not be phosphorylated andincorporated into virus nucleic acid, owing tode novo thymidine 5'-triphosphate productionand a low-level host cell thymidine kinase

gene. By adding aminopterin, host cell thymi-dine kinase activity rises (16), and one wouldthen expect inhibition by BUdR. Aminopterinplus BUdR did inhibit the AL-5 isolate (3.71log10), whereas aminopterin plus thymidine didnot, indicating that aminopterin alone does notaffect viral replication. Consequently, it is sus-pected that the isolates are deficient in thymi-dine kinase activity, but this assumption war-rants further investigation. Differences in thy-midine kinase-inducing ability have previouslybeen noted for certain members of the herpesvi-rus group (17).

In summary, these two capuchin monkey vi-rus isolates appear to be new additions to theherpesvirus group. Furthermore, the two vi-ruses are species specific, since neutralizing an-tibodies to the isolates were detected only incapuchin monkey sera.

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