INFETMION AND IMMUNITY, July 1978, p. 129-1340019-9567/78/0021-0129$02.00/0Copyright X 1978 American Society for Microbiology

Vol. 21, No. 1

Printed in U.S.A.

Persistent Shedding of Adenovirus in Urine of ChimpanzeesDAVID M. ASHER,* JOHN J. HOOKS,t HERBERT L. AMYX, NANCY P. LUBER, LUDMILA V.

SHAVRINA ASHER,t CLARENCE J. GIBBS, JR., AND D. CARLETON GAJDUSEK

Laboratory of Central Nervous System Studies, National Institute ofNeurological and CommunicativeDisorders and Stroke, National Institutes ofHealth, Bethesda, Maryland 20014

Received for publication 7 November 1977

A new adenovirus, designated Pan 11, was isolated repeatedly from the urineof several chimpanzees for more than 1 year. One chimpanzee had chronicinterstitial nephritis; the others were healthy. Most chimpanzees tested hadneutraliig antibodies to Pan 11 virus in the serum; three people who workedwith chimpanzees also acquired antibodies. Transplantable rhabdomyosarcomasdeveloped in hamsters inoculated as newborns with Pan 11 virus.

A search for new simian cytomegaloviruses (2)led to the unexpected finding that many appar-ently healthy chimpanzees in a research colonyhad adenoviruses in their urine (D. M. Asher, J.Hooks, H. Amyx, H. Barki, N. Luber, C. J.Gibbs, Jr., and D. C. Gajdusek, Abstr. Annu.Meet. Am. Soc. Micribiol. 1974, V74, 213). Wedescribe here an adenovirus that has been iso-lated repeatedly from the urine of chimpanzees.We also present results of a serological studywhich indicates that the virus is widespreadamong the chimpanzees and that people in closecontact with chimpanzees have been infected.

MATERIALS AND METHODSAnimals. Juvenile chimpanzees, purportedly from

West Africa, were purchased from importers. Theprimate colony has been described (1). Animals werein the colony from 7 days to 6 years when first studied.Six chimpanzees had previously been inoculated withmaterials containing the agents ofkuru or Creutzfeldt-Jakob disease; 10 others were uninoculated at thebeginning of the study but were subsequently injectedwith those or other materials.Virus isolations. Urine samples were obtained by

bladder puncture as previously described (2), and allspecimens were prepared by modification of standardtechniques for virus isolation (32) in human diploidfibroblast cultures.

Cell cultures WI38 human diploid fibroblast cul-tures, rhesus monkey kidney cells, African green mon-key kidney cells, rabbit kidney cells, and BHK 21hamster kidney heteroploid cells were purchased fromFlow Laboratories, Inc., Rockville, Md., and MA184human diploid fibroblasts, human embryo kidney(HEK) cells, and MA117 African green monkey lungfibroblasts were purchased from Microbiological As-sociates, Bethesda, Md. HeLa cells were obtained fromL. Horta Barbosa of the National Institute of Neuro-logical and Communicative Disorders and Stroke, Na-

t Present address: Laboratory of Oral Medicine, NationalInstitute of Dental Research, National Institutes of Health,Bethesda, MD 20014.

tional Institutes of Health (NIH), Bethesda, Md., andCV1 African green monkey kidney heteroploid cellswere from A. Lewis of the National Institute ofAllergyand Infectious Diseases (NIAID), NIH. Brain cellsfrom chimpanzees, squirrel monkeys, and owl monkeysand rhesus monkey lung fibroblasts were derived inour laboratory. Cells were grown in standard gastubes, plastic flasks, or 24-well trays (Linbro ScientificCo., Hamden, Conn.). Explant cultures were preparedas previously described (31). All cells were grown inEagle minimal essential medium in Earle salt solutionwith 10% heat-inactivated fetal calf serum and 50 igof gentamicin and 30 gAg of L-glutamine per mL Diploidand heteroploid cells were maintained in the samemedium with 5% and 2% calf serum, respectively.Fluids were changed twice weekly.

Characterization of viru8. Electron microscopicexamination of glutaraldehyde-fixed infected cell cul-tures was performed by Meloy Laboratories, Inc.,Springfield, Va. Hemagglutination tests were per-formed by standard methods in tubes and microtiterU-bottom trays (28), using 0.85% NaCl as the diluentand 0.5, 0.8, and 1.0% erythrocyte concentrations.Erythrocytes known to be agglutinated by adenoviruswere obtained from rhesus monkeys and Wistar rats.Human adenovirus type 7 (Flow Laboratories) servedas the control. Human type 0 and guinea pig eryth-rocytes were also tested. All reactions were carried outat 37, 22, and 40C. Hemagglutination tests were alsocarried out with rat cells in the presence of 5% rabbitantiserum to human adenovirus type 5 (33).

Serological tests. Complement fixation and neu-tralization tests were performed as previously de-scribed (2). Viruses for neutralization tests were fil-tered through 0.45-nm filters. When simultaneous con-trol titrations showed that there was from 10 to 10050% tissue culture infectious doses (TCID50) of virusin the test, results were scored.Sera Antiserum to one of the isolates (A-226-1)

was prepared in rabbits inoculated intravenously withvirus and intramuscularly with virus plus Freund com-plete adjuvant. Adenovirus complement fixationgrouping serum was purchased from MicrobiologicalAssociates. Neutralizing antiserum to chimpanzee ad-enovirus C-1, Bertha strain (30), was provided by J.

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Hartley, NIAID. Antisera to chimpanzee adenovirusesPan 5, Pan 6, Pan 7, and Pan 9 (4) and Pan 10 (anunpublished chimpanzee adenovirus isolated by N. G.Rogers and M. Basnight of our laboratory) were pre-

pared by the Public Health Research Institute of NewYork, Inc., Otisville, N.Y. Reference antisera to simianadenoviruses SV-1, SV-11, SV-15, SV-17, SV-20, SV-23, SV-25, SV-30, SV-31, SV-32, SV-33, SV-34, SV-36,SV-37, SV-38, SV-39, SA-7, SA-17, V-340, CV-2/M-2,and M-3 were provided by R. Heberling, SouthwestFoundation for Research and Education, San Antonio,Tex., and antiserum to SA-18 was provided by J. Duff,National Cancer Institute, NIH. Reference antisera tohuman adenovirus types 1 through 31 came from theResearch Resources Branch, NIAID, and antisera tohuman adenovirus types 20 and 21 and to candidatevirus types 32 and 33 were provided by W. Rowe,NIAID. Antiserum to candidate virus type 34 (17) wasnot yet available. All typing sera were prepared inrabbits. Chimpanzee sera were from animals in our

colony, from a colony at the Johns Hopkins UniversitySchool of Hygiene and Public Health, Baltimore, Md.,and from 19 chimpanzees newly received from Africaby an importer; monkey sera were from animalshoused in separate rooms in our colony. Human serawere from members of our staff, staffs of other labo-ratories, families of American hospital patients, andfrom several populations previously described (7); serafrom Turkey were collected in 1955, those from Ma-laysia were collected in 1958, and all others were

collected in or after 1970.Tests oftumorigenicity. Newborn hamsters were

inoculated subcutaneously over the shoulders with0.05-ml volumes of suspensions of virus or tumor cells.Control animals were inoculated with comparable sus-

pensions of frozen-thawed uninfected human diploid

fibroblast cells or with freshly prepared spleen andliver cells of normal hamsters. When their tumorsbecame very large, hamsters were exsanguinated, anddissected, and their carcasses were fixed in 10% form-aldehyde in saline. Histological examinations of thetumors were performed by R. L. Schueler of the Fred-erick Cancer Research Center, Frederick, Md., and H.Casey of the Armed Forces Institute of Pathology,Washington, D.C.

RESULTSVirus isolations from urine. Forty-nine ur-

ine specimens from 16 chimpanzees were studied(Table 1). The first two viruses were isolatedfrom urines obtained at necropsy from chimpan-zees (A-173 and A-217) with experimentalCreutzfeldt-Jakob disease. Both viruses pro-

duced patches of round, refractile cells in MA184cultures, first detected 12 days after inoculation.Cytopathic effect (CPE) progressed slowly, in-volving less than 75% of the cell sheet by the endof week 4 in culture. The titer of the secondvirus was 1035 TCID5o/ml of urine. The thirdviral isolate came from the urine of a healthyuninoculated female chimpanzee (A-226); focalareas of cellular changes similar to those pro-

duced by the first two viruses were noted incultures 7 days after inoculation, and CPE pro-

gressed rapidly, involving most cells of the mon-olayers by day 14. This isolate, designated A-226-1, was selected for further study. Its finaltiter was 1044 TCID5o/ml of urine.Cytopathogenic agents were detected at least

once in the urines of 12 of the 16 chimpanzees

TABLE 1. Viruses isolated from urines of chimpanzeesDate of specimen (yr and quarter) and result

Specimens yield-Chipanzee no. Yr of arrival 1973 1974 1975 1976 ing virus/total

specimensI II III IV II III IV IV

A-49 1966 0/1A-84 1968 - 0/1A-138 1969 - 0/1A-173 1970 +b 1/1A-201 1971 - 0/1A-217 1972 +C 1/1A-226 1972 +C + + +C - 4/5A-243 1973 +d - + - - 2/5A-244 1973 +b - - 1/4A-249 1973 - - + +b 2/4A-250 1973 +b + + + b + 5/5A-251 1973 +C +C - + - 3/6A-274 1973 - +C + _ 2/4A-275 1974 + +C - 2/3A-276 1974 + + + - 3/4A-277 1974 - + - 1/3

a +, Urine yielding cytopathogenic agent; -, urine yielding no cytopathogenic agent.b Agent lost in passage.c Agent neutralized by rabbit antiserum to A-266-1 (first isolate from chimpanzee A-226).d Agent not neutralized by rabbit antiserum A-266-1.

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ADENOVIRUS IN URINE OF CHIMPANZEES 131

studied (Table 1). From 10 animals, urine wasobtained on more than one occasion; all 10yielded a virus in at least one specimen. Allviruses produced similar CPE in human diploidfibroblast cultures after 7 to 55 days. Threeanimals had viruria demonstrated repeatedly for16 to 27 months. The identity of other isolateswith the A-226-1 strain by serum neutralizationtest is shown in Table 1.Characterization of Pan 11 virus (A-226-

1 isolate). The A-266-1 isolate grew well inhuman diploid fibroblast cultures. When undi-luted stocks of virus were passaged into freshcultures, CPE appeared in 2 to 4 days and pro-gressed to involve most cells by day 7. Stocks ofvirus, prepared by freezing cells and fluids ofcultures showing complete CPE, contained from105 to 107 TCID5o/ml.Nuclei of human diploid fibroblasts infected

with the A-226-1 isolate several days earlier andstained with acridine orange showed flecks ofbright-green material (DNA) not seen in con-

trols. Thin sections of infected cells, examinedby electron microscopy, revealed intranuclearparticles typical of adenoviruses, 80 to 90 nm indiameter with dense cores. No satellite virusparticles were found. Identification of the virusas an adenovirus was confirmed by the comple-ment fixation reaction with standard groupingserum.Serological identification. Adenovirus

group reference antiserum fixed complementwith 4 U of adenovirus 7 antigen at a serumdilution of 1:8 and with 4 U of A-266-1 isolate ata serum dilution of 1:16. Known negative serumand antigen controls were not reactive. (Sera ofchimpanzees A-226 and A-227 fixed complementwith adenovirus 7 antigen at serum dilutions of1:64 and 1:32, respectively, and both fixed com-plement with A-266-1 at dilutions greater thanor equal to 1:64.)

Dilutions of 1:10 of reference antisera to 33types of human adenovirus, 6 types of chimpan-zee adenovirus, and 22 types of monkey adeno-virus failed to neutralize 100 TCID50 of the A-266-1 isolate (Table 2). A monotypic rabbit an-tiserum prepared with A-266-1 isolate neutral-ized the same dose of homologous virus at adilution of 1:320, and serum from chimpanzee A-226 itself neutralized at a dilution of 1:1,280. Theisolate was therefore considered to be a newvirus type, designated Pan 11 by the conventionof our laboratory (27).

Neutralization tests were performed with 7 of27 isolates besides the A-226-1 isolate by titrat-ing each in the presence and absence of a 1:10dilution of the Pan 11 antiserum (Table 1). Be-tween 103 and 105 TCID50 of 6 strains were

TABLE 2. Chimpanzee adenovirus Pan 11:neutralization tests with antisera

Reciprocal ofSera neutralization

titer'

Infected chimpanzeeb ................... 1,28062 Monotypic reference antiseracChimpanzee adenovirus Pan j1d ....... 32033 Human adenovirus types' ........... <106 Chimpanzee adenovirus types( ....... <1022 Monkey adenovirus types .......... <10a Neutralization titer = serum dilution inhibiting

100 TCID5o of virus.b Chimpanzee A-226.¢ Sera prepared in rabbits.d A-226-1 strain.'Types 1 through 6, 7A, 8 through 31, and candidate

adenoviruses 32 and 33.fTypes C-1, Pan 5 through 7, Pan 9, and Pan 10.' Types SV-1, SV-11, SV-15, SV-17, SV-20, SV-23,

SV-25, SV-30 through -34, SV-36 through -39, SA-7,SA-17, SA-18, V-340, CV-2/M-2, and M-3.

neutralized. The 7th strain, tentatively identifiedas an adenovirus by its typical CPE in W138 andHEK cells, was not neutralized, but it was notfurther identified since it grew poorly on subse-quent passage. Neutralization tests could not beperformed with 5 other isolates lost in passage.Fifteen other isolates were not tested. The 4thisolate from the urine of chimpanzee A-226 wasneutralized by antiserum to Pan 11 virus. In all,7 isolates from five chimpanzees were success-fully neutralized by antiserum prepared with theoriginal A-266-1 strain of Pan 11 virus.Hemagglutination tests. Preparations of

Pan 11 virus containing more than 105TCID5o/ml failed to agglutinate all concentra-tions of rhesus monkey and rat erythrocytestested after incubation at 37, 22, and 40C. Therewas also no agglutination with human type 0 orguinea pig erythrocytes. Some types of humanadenovirus agglutinate rat erythrocytes only inthe presence of antiserum to a heterotypic ade-novirus of Rosen group 3 (33); however, raterythrocytes were not agglutinated by Pan 11virus diluted in 5% antiserum to human adeno-virus type 5.Host range. CPE was produced by the A-

266-1 isolate in human, chimpanzee, rhesus mon-key, and African green monkey diploid cells, butnot in HeLa or CV1 cells, observed for 30 days.Squirrel monkey and owl monkey brain cells,primary rabbit kidney cells, and BHK21 cellsalso showed no CPE. No lesions in membranesor embryos of hens' eggs inoculated on thedropped chorioallantoic membrane were ob-served 48 and 72 h later. No illnesses occurredin newborn mice or hamsters inoculated intra-

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cerebrally on day 1 of life. Subcutaneous inoc-ulation of hamsters produced tumors.Tumors in hamsters. Fifty-five of 80 ham-

sters inoculated subcutaneously as newbornswith Pan 11 virus developed undifferentiatedspindle cell tumors in 3.5 to 10.5 months; whencultured cells of one such tumor were inoculatedinto newborn or weanling hamsters, well-differ-entiated embryonal rhabdomyosarcomas devel-oped rapidly. These findings will be described indetail elsewhere.Pathogenesis ofPan 11 adenovirus infec-

tion. Only one chimpanzee had signs of renaldisease. Until clinical signs of kuru or Creutz-feldt-Jakob disease appeared, all other animalswere healthy. Urine specimens had specific grav-ities between 1.001 and 1.007, pH 5 to 6.5, andno ketones or protein. An occasional urine con-tained a small amount of glucose, presumablydue to the intravenous infusion. There were rarelarge nucleated cells in spun urine sediment.Stained preparations of sediment were spoiledby heavy precipitates of mannitol. Blood ureanitrogen and serum creatinine contents werewithin the normal human range (<8 and <1.0mg/dl, respectively). Seven of 16 chimpanzeesdied after the study began, all except 1 of kuruor Creutzfeldt-Jakob disease. One animal wasfound to have severe diffuse interstitial nephri-tis; that animal, however, had suffered severaltransient episodes of intravascular hemolysis,hemoglobinuria, and elevated blood urea nitro-gen, probably due to ingestion of a phenolicdisinfectant which was demonstrated in its tis-sues. Kidneys, ureters, and bladders of otheranimals appeared normal.Pan 11 virus was isolated only from the urine

of chimpanzees. Explant cultures of kidneys of3 animals and of other urinary tract tissues of 1were attempted, though not all grew success-fully; in no case was CPE observed during sev-eral months of cultivation. Suspensions of kid-ney tissue from 10 animals and of ureter, blad-der, and urethra from 1 yielded no cytopathicagent in HEK and W138 cell cultures. Attemptswere made to isolate virus from blood, stool, andpharyngeal secretions of chimpanzees. Agentsproducing syncytia in HEK cultures were iso-lated from buffy coat leukocyte preparations ofthe first 3 animals studied; lysed whole bloodfrom 1 yielded no virus. Throat swabs from 4 of10 animals also yielded syncytium-formingagents, whereas fecal swabs of 4 of 9 chimpan-zees contained agents causing rounding of cellsthroughout cultures. The first three syncytium-forming agents from buffy coats were identifiedby neutralization with monospecific rabbit seraas simian foamy virus types 6 (two isolates) and7. The isolates from throat and rectal swabs,

none of which gave CPE resembling that ofadenovirus, were not identified.Serum survey. Sera obtained in 1973 and

1974 from 31 chimpanzees in our colony allneutralized 100 TCID5o of Pan 11 virus at serumdilutions of 1:10 or greater (Table 3). Sera col-lected from 8 of the same chimpanzees 7 to 11years earlier were also tested; 7 already hadneutralizing antibodies in the earliest availableserum, including a serum taken in August 1963,shortly after the colony was begun. One chim-panzee had no neutralizing antibodies in seracollected during its first 6 months in the colony,whereas its sera contained antibodies at 9months and thereafter. Sera of 6 chimpanzeeswere tested in serial dilution; they neutralizedPan 11 virus at dilutions of 1:160 to 1:>2,560(geometric mean titer, 2654). The sera of 4animals from another colony all neutralized thevirus. Seventeen of 19 sera from chimpanzeesnewly received from West Africa also containedneutralizing antibodies. Sera of 28 monkeys ofthree old-world species (8 rhesus, 5 cynomolgusand 8 African green monkeys) and three new-world species (1 squirrel, 3 capuchin, and 3 spi-der monkeys) were tested at dilutions of 1:5 or1:10; none neutralized the virus. (These animalshad no contact with chimpanzees.)Sera from 112 people of various populations,

including 34 members of our staff, were testedat serum dilutions of 1:5 or 1:10 (Table 4). Threesera, all from animal caretakers in our primatecolony, neutralized Pan 11 virus; sera from 12other full-time animal caretakers and from 11other people who worked frequently with chim-panzees or Pan 11 virus had no demonstrableantibodies. Sera of 33 other North Americanadults, and smaller numbers of South Ameri-cans, Africans (Nigerian city dwellers), Turks,and Malaysians also failed to neutralize Pan 11virus. Sera collected throughout the terms ofemployment of the 3 seropositive animal care-takers were tested in serial dilutions. Serumfrom year 1 of employment of 1 caretaker couldnot be found; year-i sera of the other 2 men hadno antibodies. All later sera neutralized Pan 11

TABLE 3. Neutralizing antibodies to adenovirusPan 11 in sera of chimpanzees

SeraChimpanzee group No: P

No. testedtive'

Newly arrived from Africa 17 19Colony rearedSera obtained after 1972 31 31Sera obtained before 1968 7 8a Neutralizing 100 TCID50 of Pan 11 virus at a serum

dilution of 1:10.

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TABLE 4. Neutrlizing antibodies to adenovirusPan 11 in human sera

SeraPopulation

No. positive' No. testedU.S.A.Animal caretakers 3 15Others in contactwith chimps 0 11

or Pan 11 virusOther adults 0 33

Nigeria 0 20Equador-Colombia 0 16Malaysia 0 9Turkey 0 8

a Neutralizing 30 to 100 TCIDso of Pan 11 virus ata serum dilution of 1:10 or less.

virus at dilutions of 1:64 or greater. The onlyman still working with chimpanzees was in goodhealth, and no virus was isolated from his urineon two occasions.

DISCUSSIONAdenovirus Pan 11 failed to agglutinate eryth-

rocytes of rats or monkeys, which places it ingroup IV ofthe classification adapted by Rapoza(26) from the system generally used'for humanadenoviruses (29). Pan 11 was not neutralized bymonotypic reference antisera to 33 types of hu-man adenovirus, 6 types of chimpanzee adeno-virus, and 22 types of simian adenovirus (includ-ing SA-7, the only known member of simianhemagglutination group IV) and is presumablya new serotype. Many other adenoviruses havebeen isolated from stools and tissues of nonhu-man primates (16), including several types fromchimpanzees. Most of the adenoviruses previ-ously isolated from chimpanzees are antigeni-cally related to known human types (18, 19, 30,31), but a few have been entirely novel (4); manyremain unstudied (35). We are not aware ofother isolations of adenovirus from urine of non-human primates, though adenoviruses havebeen recovered from cultures of monkey kidneycells (16). Adenoviruses have not been isolatedfrom cultures ofchimpanzee kidney cell cultures,not even from kidneys of animals whose urinewas found to contain virus at the time of death.Not all viruses isolated from the urine of chim-panzees were identified as adenovirus Pan 11,and it is possible that some may be other ade-noviruses.

Infection with Pan 11 virus is widespreadamong captive chimpanzees. It appears to haveinfected most chimpanzees before their arrivalin our colony and has been present since thecolony began. Infection of some animals proba-bly occurred in the wild, although it might havetaken place during quarantine before exportfrom Africa. There is no evidence that primates

besides chimpanzees and humans have been in-fected. Three animal caretakers acquired anti-bodies to Pan 11 virus within 2 years of workingwith chimpanzees. Sera of 109 other people con-tained no antibodies. Only one other "simian"adenovirus, SV-20, has been previously reportedto infect humans (3). Antibodies to SV-20 werefound in the sera of a substantial number ofpeople, most ofwhom had no contact with mon-keys; therefore SV-20 may be a natural adeno-virus ofhumans as well as simians. Pan 11 virus,however, seems to be native to chimpanzees.Pan 11 virus was excreted persistently for

more than 1 year in the urine of three animalin spite of circulating antibodies. It was isolatedonly from the urine and not from the blood,secretions, or organs, although several other ad-enoviruses have been easily recovered fromlymph nodes ofchimpanzees (4). Similar chronicurinary excretion of adenoviruses has been oc-casionally observed in humans (5, 14) and is acommon phenomenon in mice (9, 15) and dogs(13, 25). The relationship between such chronicadenoviral infections and diseases of the urinarytract is not clear (36). Severe disseminated ade-novirus infections in hmans may be accompa-nied by clinical and histological signs of intersti-tial nephritis (8, 34), and adenoviruses have beenisolated from urines of patients with mild aden-oviral respiratory illnesses (11, 12), as well asfrom urines and kidneys of patients with fataldisseminated disease (6, 17). However, adenovi-ruses have also been found in kidneys of patientswithout evidence either of systemic adenoviralinfection or of overt renal disease (20, 22). Theonly disease of the human urinary tract in whichadenoviruses have been convincingly implicatedis acute hemorrhagic cystitis (21, 23, 24). Miceand dogs with persistent adenovirus infectionsof the kidneys have chronic diffuse interstitialnephritis (9, 37) and are more susceptible tobacterial infections of the urinary tract than arenormal animals (9, 10). Chimpanzees seem gen-erally to tolerate their chronic adenoviral infec-tions of the urinary tract without signs of dys-function. However, in chimpanzees, and proba-bly other primates, chronic infection of the uri-nary tract may be a reservoir for adenovirus, andurine may be a vector.

ACKNOWLEDGMENTSWe thank Alfred E. Bacote, Michael P. Sulima, and Gerald

Murphy for technical assistance.

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