Isolation of a New Simian Foamy Virus from a Spider Monkey Brain Culture

Transcription

1 INFECTION AND IMMUNITY, Nov. 1973, p Copyright American Society for Microbiology Vol. 8, No. 5 Printed in U.S.A. Isolation of a New Simian Foamy Virus from a Spider Monkey Brain Culture JOHN J. HOOKS, C. J. GIBBS, JR., S. CHOU, R. HOWK, M. LEWIS, AND D. C. GAJDUSEK National Institute of Neurological Diseases and Stroke, Bethesda, Maryland 20014, West Virginia University Medical Center, Morgantown, West Virginia 26506, and Meloy Laboratories, Springfield, Virginia Received for publication 2 July 1973 A syncytium-forming (foamy) virus was isolated from a spider monkey brain cell culture. Cytopathic effect was observed both in the brain culture and in human embryonic kidney cells. Neutralizing antibody was present in the sera of the spider monkey from whom the isolation was made. The virus was inhibited by 5-bromo-2-deoxyuridine (20 utg/ml), contained a ribonucleic acid-dependent deoxyribonucleic acid polymerase, and had an infectivity peak at 1.15 g/cm3 in a sucrose density gradient. The virus passed through a 220-nm but not a 100-nm membrane filter, was chloroform sensitive, and was inactivated at 56 C in 30 min. Hemagglutinating and hemadsorption activity was not noted with a variety of erythrocytes. The virion was spherical, formed in the cytoplasm, and was 105 to 115 nm in diameter. Ring-shaped nucleoids, 45 to 50 nm in diameter, were associated with tubular profiles. The virus was not neutralized by sera prepared against known viruses, including simian foamy virus types 1 through 7, Mason-Pfizer monkey virus, and bovine syncytial and measles viruses. Sera from a rabbit hyperimmunized with the isolate and sera from 19 spider monkeys had neutralizing antibody to the isolate; however, these sera did not cross-react with simian foamy virus types 1 through 7. Neutralizing antibody to the isolate was not detected in sera from 16 humans, 9 rhesus monkeys, and 10 chimpanzees. Viruses isolated from a number of primate at necropsy. The tissue was placed in Eagle minimal species, which have as one of their characteristics the induction of syncytia formation in cell U/ml), streptomycin (100 ug/ml), and 20% fetal essential medium supplemented with penicillin (100 cultures, have been referred to as the simian bovine serum inactivated at 56 C for 60 min. Explant tissue cultures were prepared according to the technique described by Rogers et al. (16). These cultures, foamy viruses. Viruses which induce syncytia and have biological properties similar to the incubated at 35 C in an atmosphere containing 5% simian viruses have also been isolated from CO2, were observed and fed at approximately 4-day bovine and feline species (10, 11, 14). The intervals. When the outgrowth from the explant tissue viruses in this group, referred to in the literature fragments was about three-fourths confluent, the cells as syncytium-forming viruses, contain ribonucleic acid (RNA), are chloroform sensitive, have 0.02% ethylenediaminetetraacetic acid and subcul- were treated with a mixture of 0.05% trypsin and a buoyant density of approximately 1.16 g/ml, tured into new flasks. and have an RNA-dependent deoxyribonucleic Virus isolation and passage. A cytopathic effect acid (DNA) polymerase (7, 14, 15). In their (CPE) was noted in primary explant cultures and undiluted supernatant fluids from these explant cultures induced a syncytial CPE in human embryo natural host, they persist in a number of tissues in the presence of neutralizing antibody (5-7, kidney (HEK) cell cultures. HEK tissue culture 16) stationary tubes were used for preparation of virus Ṫhis study describes the isolation, characteristics, and distribution of a new foamy virus Other viruses used in this study were: simian foamy pools and for viral infectivity titrations. isolated from a spider monkey brain culture virus types 1 through 5, obtained from the National grown in vitro. The characteristics are described Cancer Institute Reference Reagent Branch; types 6 and in relation to the properties of the 7, isolated from chimpanzees in our laboratory syncytiumforming group of viruses. NIH strain); and vesicular stomatitis virus (Indiana (6); vaccinia virus (Division of Biologics Standards, MATERIALS AND METHODS strain). Tissue culture and media. Tissue cultures used in Explant culture preparation. Cerebral cortical viral susceptibility studies were: HEK, human skin tissue of a spider monkey (S-140) brain was obtained and muscle (MA-337), human fetal brain (FL-3000), 804

2 VOL. 8, 1973 NEW SPIDER MONKEY FOAMY VIRUS 805 Vero (African green monkey kidney), and human to the method of Ross et al. (18) through the Sephadex sarcoma (MA-160). Tissue cultures were obtained G-100 step without the phase extraction step. from Microbiological Associates Inc., Bethesda, Md. Density gradients. HEK cell cultures grown in Low-passage MA-160 cells were kindly supplied by stationary tubes were infected with spider monkey Monroe Vincent of Microbiological Associates. foamy virus. When approximately 30 to 40% of the HEK tissue cultures were maintained with Eagle cells showed CPE, the cells were subcultured into basal medium, prepared with Earle salt solution, and flasks. After an additional 48 h of incubation, supernatant fluids were collected and centrifuged at 35,000 contained 100 U of penicillin, 100 gg of streptomycin per ml, and 2 to 5% fetal bovine serum inactivated at rpm for 2 h. Virus pellets were resuspended in 56 C for 60 min. one-tenth of original volume in Eagle medium. Resuspended pellets were layered over a 10 to 50% sucrose Antisera. Spider monkey foamy virus, 11th HEK passage, was clarified at a maximal relative centrifugal force of 930 x g for 30 min, and resultant Fractions were collected from the bottom of the tubes, gradient (wt/vol) and spun at 25,000 rpm for 160 min. supernatant fluids were used for preparation of specific antisera. A white New Zealand rabbit was bled cell cultures. and serial 10-fold dilutions were inoculated into HEK and inoculated with viral suspensions mixed with an Electron microscopy. For electron microscopy equal volume of Freund complete adjuvant. The studies, virus-infected and control HEK cell cultures virus-immunizing dose was followed 25 days later by a were harvested with a rubber policeman. The suspended cells were pelleted by centrifugation, fixed in 2-ml virus-adjuvant boostering dose by the subcutaneous and intraperitoneal routes. The rabbit was bled 2.5% gluteraldehyde for 30 min, and suspended in on days 38 and 46. Millong buffer. The cells were postfixed in 1% buffered osmium tetroxide for 1 h, dehydrated in graded Reference-specific antisera were used to determine the viral relationship of the spider monkey foamy ethanol, and embedded in an epoxy resin (Epon-Araldite) mixture. Ultrathin sections stained with uranyl virus to other syncytial-producing viruses and other viruses by neutralization tests. Antisera to simian acetate and lead citrate were examined with an foamy virus types 1 through 5 were supplied by electron microscope (AEI-Corinth). National Cancer Institute Reference Reagent Branch, Serology and epidemiological survey. Neutralization tests with spider monkey foamy virus and the and antisera to types 6 and 7 were prepared by us in rabbits (6). Antisera to respiratory syncytial virus, simian foamy virus types 1 through 7 were carried out parainfluenza types 1, 2, and 3, SV5, and measles and as previously described (6). Human serum was collected from personnel who routinely conduct necropsy mumps viruses were obtained from Microbiological Associates, Bethesda, Md. Antisera to the following procedures on primates and from our animal caretakers handling numerous primate species. Spider herpesviruses were kindly supplied by L. Melendez, New England Regional Primate Research Center, monkey, chimpanzee, and rhesus monkey sera were Southborough, Mass.: spider monkey herpes, herpes collected from animals housed in our own facilities. ateles, sand rat nuclear inclusion agent, herpes aotus types 1 and 2, owl monkey kidney isolate, herpes RESULTS simplex, herpes-b, herpes saimiri, and herpes-t. Antisera to bovine syncytial virus was kindly supplied Syncytia of the type usually associated with by M. Van Der Maaten, National Animal Disease simian foamy viruses were observed in spider Center, Ames, Iowa. monkey (S-140) cerebral cortex explant and Physical and chemical characteristics. Viral sensitivity to 5-iodo-2-deoxyuridine (IUdR) and to 5- from these monkey brain cultures induced syn- second-passage cultures. Supernatant fluids bromo-2-deoxyuridine (BUdR) was determined by the cytia in HEK cells after 20 days of incubation. method described by Parks and Todaro (14). Viral Only undiluted supernatant fluids induced sensitivity to chloroform treatment, filtration techniques, and hemagglutination and hemadsorption CPE in the first three virus passages in HEK techniques have previously been described cells. (4, 17, 23). Adaptation of the virus to growth in HEK RNA-dependent DNA polymerase assay. Spider cells occurred in subsequent passages and was monkey foamy virus was concentrated from culture associated with a reduced incubation period media by centrifugation at 100,000 x g through 20% and increased infectivity titer. Additional glycerol. The high-speed pellet was tested for DNA strains of spider monkey foamy virus have polymerase activity by using a variety of templates. recently been isolated from explant cultures of Assay with activated DNA was carried out as described by Ross et al. (18). Assays using the templates Syncytia were noted in these primary explant spider monkey liver, spleen, and brain tissues. poly(ra).oligo(dt) and poly(rc).oligo(dg) were conducted as described by Ross et al. (18) and Scolnick et cultures 3 to 9 weeks after incubation. In an al. (20). Poly(rA) was obtained from Miles Laboratories, Kankakee, Ill., and poly(ra) oligo(dt) and host range in vivo and in vitro, mice, hamsters, attempt to evaluate the experimental poly(rc) oligo(dg) were obtained from Collaborative and various cell lines were used. Newborn mice Research, Waltham, Mass. The template specificity and hamsters, inoculated by the intracerebral of the foamy virus polymerase activity was compared and subcutaneous routes, failed to develop with that of woolly type-c virus reverse transcriptase clinical signs of disease when observed for 1 (20). The woolly virus enzyme was prepared according year. In contrast, the virus induced characteris-

3 806 HOOKS ET AL. INFECT. IMMUNITY tic CPE on first passage in HEK cell cultures and in human skin and muscle (MA-337), human fetal brain (FL-3000), human sarcoma (MA-160), and Vero cells. Sensitivity to base analogues. The effects of IUdR and of BUdR on the replication of the spider monkey foamy virus are shown in Tables 1 and 2. At a concentration of 10-4 M, IUdR inhibited spider monkey foamy virus replication by only 0.5 logs, whereas vaccinia virus was inhibited by 2.1 logs. The addition of thymidine reversed the effect of IUdR. Vesicular stomatitis virus, a control RNA-containing virus, was not inhibited by IUdR. Spider monkey foamy virus replication was inhibited by 0.5, 1.7, and 2.2 logs by the addition of BUdR at a final concentration of 10, 20, and 50 mg/ml, respectively (Table 2). Vaccinia virus was inhibited 2.3 logs by BUdR at a concentration of 20,ug/ml. The inhibitory effect of BUdR was reduced by the addition of thymidine. Vesicular stomatitis virus was not inhibited by BUdR at concentrations of 20 or 50 ;Lg/ml. DNA polymerizing activity. The high-speed pellet derived from supernatant culture media TABLE 1. Sensitivity of spider monkey foamy virus and control viruses to IUdRa Log Virus Treatment Titer inhibition Spider monkey foamy IUdR IUdR + thy midine None 2.4 Vaccinia IUdR IUdR + thy midine None 6.2 Vesicular stomatitis IUdR IUdR + thy midine None 7.2 a Three human embryonic kidney cultures were inoculated with spider monkey foamy virus, vaccinia virus, or vesicular stomatitis virus (VSV). After a 2-h absorption, inocula were removed and IUdR alone (10-4 M), IUdR plus thymidine (10-3 M), or media were added. After 19 h (VSV and vaccinia virus) and 24 h (spider monkey monkey foamy virus), the fluids were removed, the cells were washed three times, and fresh medium was added. At 28 h (VSV and vaccinia virus) and 72 h (spider monkey foamy virus) postinoculation, virus was harvested by freezing and thawing. Infectivity titrations were performed in HEK cell cultures and are expressed as log10 TCD5/ ml. of the spider monkey foamy virus-infected HEK cells contained DNA polymerase activity (Fig. 1) with different templates. The woolly monkey type-c virus was used as a positive control. The foamy virus polymerase activity exhibited a marked preference for the synthetic template poly(ra) -oligo(dt) when compared with activated DNA similar to the woolly type-c virus RNA-dependent DNA polymerase. Both systems utilized poly(rc) oligo(dg), although less well than with either poly(ra) oligo(dt) or activated DNA similar to previous reports (20). Cellular DNA polymerases, on the other hand, utilized activated DNA preferentially and had almost no activity with poly(rc).oligo(dg). Viral characteristics. The concentrations of spider monkey foamy virus in various fractions collected from virus on a sucrose gradient (10 to 50%) are shown in Fig. 2. A distinct peak of infectivity was noted in fraction 9, which had a density of 1.15 g/cm3. Chloroform treatment decreased the infectivity of spider monkey foamy virus by 2.4 log10. Simian foamy virus type 7, a chloroform-sensitive control virus, was decreased by 3 log1o, whereas adenovirus, a chloroform-resistant control, was not affected by chloroform treatment. When the spider monkey foamy virus was held at 0 or 37 C for 30 min, the viral infectivity remained constant at mean tissue culture TABLE 2. Sensitivity of spider monkey foamy virus and control viruses to BUdRa Spider mon- Vaccinia Vesicular key foamy virus stomatitis virus Conc BUdR (qg/ml) Log Log Log Titer inhi- Titer inhi- Titer inhibition bition bition No drug BUdR NTb NT BUdR + thymidine NT 20 BUdR BUdR + thymidine BUdR NT BUdR thymidine a The experiment was done as described in Table 1, except that BUdR alone was added at 10, 20, and 50 mg/ml, and that, at 28 h (VSV) and 48 h (vaccinia and spider monkey foamy virus) postincubation, virus was harvested by freezing and thawing. b NT, Not tested.

4 VOL. 8, 1973 NEW SPIDER MONKEY FOAMY VIRUS HI SPIDER MONKEY WOOLY MONKEY FOAMY VIRUS POLYMERASE TYPE C VIRUS POLYMERASE FIG. 1. The high-speed pellet from the S-140 virus culture media was suspended in 0.01 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (ph 7.8), 0.1 M NaCl, and M dithiothreitol (DTT) containing 0.1% Triton X-100 at 100 to 300 times the concentration of the starting material. Assays using poly(ra) oligo(dt) or poly(rc) oligo(dg) contained in a final volume of 0.05 ml: 0.04 M Tris-hydrochloride, ph 7.8; 0.06 M KCI; M DTT; M MnCl2; 0.1% Triton X-100; either 10-5 M 3H-thymidine triphosphate (TTP) or 10-5 M 3H-deoxyguanosine triphosphate (dgtp); and either 6 pg of poly(ra) per ml and 6 pg of oligo(dt) per ml or 60 pg of poly(rc) oligo(dg) per ml and the amount of enzyme designated in the figure. Assay with activated DNA contained in a final volume of 0.05 ml: 0.08 M Tris-hydrochloride, ph 7.8; 0.06 M Mg (Ac)2; M DTT; 10-4 M deoxyadenosine triphosphate, dg TP, and deoxycytidine triphosphate; 10-5 M 3H-TTP; 50 pg of activated DNA per ml prepared as described by Ross et al. (18); and the amount of enzyme indicated on the abscissa. Incubations were at 37 C for 1 h. Acid-precipitable radioactivity was counted on membrane filters in a liquid scintillation counter. Symbols: Poly(rA) oligo(dt), 0; activated DNA, 0; poly(rc) oligo(dg), A. dose (TCD50)/ml. In contrast, virus titer dropped 1.8 log10 after 30 min at 45 C, and no infectious virus could be detected in samples held for 30 min at 56 C. The spider monkey foamy virus did not hemagglutinate spider monkey, guinea pig, chicken, sheep, or human 0 red blood cells tested at 0.5, 0.8, and 1.0% concentrations and at temperatures of 4, 22, and 37 C. S-140 virus-infected HEK cells did not display hemadsorption with freshly prepared 0.5% guinea pig erythrocytes. The virus passed through a 220-nm but not through a 100-nm membrane filter (Millipore Corp.). Electron microscopy. By electron microscopy, virus particles were observed only within the cytoplasm, especially in the cells displaying multinucleation. Intracytoplasmic ring-shaped nucleoid particles measuring 45 to 55 nm in diameter often aggregate together, forming colonies with electron-dense amorphous material in the background (Fig. 3). Two morphologically distinct virions being enclosed either by single or double envelopes in their various intermediate stages of development are present. The single membrane-enveloped virions measured 80 to 85 nm in diameter and were often collected within the cytoplasmic vacuoles of the smooth endoplasmic reticulum (Fig. 4), forming their own colonies. They were frequently lined up along the wall of empty cytoplasmic vacuoles. In contrast, the double membrane-enveloped virions appeared to derive from the nucleoids which were enclosed by tubular membrane profiles, measuring 25 to 30 nm apart (Fig. 5). The tubular membrane profiles engulfing two or three nucleoids are seen to finally enclose an individual particle to form a complete virion, 105 to 115 nm in diameter (Fig. 6). The single membrane-enveloped virions are morphologically indistinguishable from those seen in the cytoplasmic vacuoles and appear interchangeable with each other. Budding forms over the protoplasmic membrane and outside spikes were not observed. E N Ci 0 o a c. z lo, 12 Top FRACTION NUMBER FIG. 2. Spider monkey foamy virus was layered onto a 10 to 50%o sucrose gradient (wt/vol). The gradient tubes were centrifuged in a Beckman SW-40 rotor at 25,000 rpm for 2.5 h. Fractions were collected from the bottom of the tube and infectivity titrations of these fractions were performed in HEK cell cultures. (12 0 In z 3 0

5 808, ^3_.,. i ; T iiu,, : 4, o 4A HOOKS ET AL. INFECT. IMMUNITY FIG. 3. Spider monkey foamy virus-infected HEK cells. Ring-shaped nucleoid particles observed in the cytoplasm. x52,000. Serological relationship of the spider monkey isolate to other viruses. Spider monkey (S-140) virus relationship to the simian foamy viruses is shown in Table 3. Serum from spider monkey S-140, from whose tissue the isolation was made, completely neutralized spider monkey foamy virus at a dilution of 1: 32. Antisera to spider monkey foamy virus prepared in a rabbit neutralized the S-140 virus at a dilution of 1:20. The S-140 spider monkey sera and the immunized rabbit sera did not neutralize approximately 100 TCD5O units of simian foamy virus types 1 through 7. In addition, S-140 virus was not neutralized by a 1: 10 dilution of antisera prepared against the seven serotypes of simian foamy viruses. Spider monkey sera, at a 1:10 dilution, were examined for the presence of neutralizing antibody to the spider monkey foamy virus isolate and to the simian foamy virus types 1 through 7 (Table 4). Neutralizing antibody to spider monkey foamy virus was detected in 5 of 11 spider

6 -VOL. 8; 1973 NEW SPIDER MONKEY FOAMY VIRUS 809 Downloaded from =..10 _o-.~ PO FIG. 4. Spider monkey foamy virus-infected HEK cells. Single membrane-enveloped particles collected within the cytoplasmic vacuoles of the smooth endoplasmic reticulum. x 75,000. monkey sera, whereas neutralizing antibody to simian foamy virus types 1 through 7 was not detected in the spider monkey sera tested at a 1: 10 dilution. One spider monkey serum, tested at a 1:4 dilution, neutralized simian foamy virus type 7. Antisera prepared against the following prototype viruses failed to neutralize approximately 100 TCD,O units of spider monkey foamy virus: bovine syncytial virus, Mason-Pfizer monkey virus, SV5, measles, parainfluenza types 1, 2, and 3, respiratory syncytial virus, mumps, herpes aotus types 1 and 2, herpes suis, owl monkey kidney isolate, herpes simplex, herpes-b, herpes saimiri, herpes-t, microtus mouse kidney isolate, and sand rat nuclear inclusion virus. In addition, antisera at a 1:4 dilution, prepared against the two spider monkey herpes viruses herpes ateles and spider monkey herpes, were devoid of neutralizing antibody to S-140 virus. Epidemiological survey for antibody to on July 7, 2018 by guest

7 810 HOOKS ET AL. INFECT. IMMUNITY - i,.bww&l.v jw,41 e :kk Ol>t I -taiee ~V i' ttv4#,,s p0 Downloaded from FIG. 5. Spider monkey foamy virus-infected HEK cells. Double membrane-enveloped particles appear to derive from nucleoids enclosed by tubular membrane profiles. x87,000. S-140 isolate. To determine the distribution of the spider monkey foamy virus in spider monkeys, other subhuman primates, and man, appropriate sera were examined for the presence of neutralizing antibody (Table 5). Neutralizing antibody to the spider monkey foamy virus was detected in the sera from 19 of 31 (61%) of the spider monkey sera tested at a 1:4 dilution. Neutralizing antibody to S-140 virus was not detected in 9 rhesus monkey or in 10 chimpanzee sera tested. Also, sera at a 1: 4 dilution from seven animal caretakers and nine laboratory personnel were devoid of neutralizing antibody to S-140 virus. DISCUSSION Simian foamy viruses have been isolated from numerous primate species, including rhesus (Macaca mulatta), cynomolgus (Macaca fasicularis), Formosan rock macaque (Macaca cyelopsis), vervet (Cercopithecus pygenythrus), on July 7, 2018 by guest

8 VOL. 8, 1973 NEW SPIDER MONKEY FOAMY VIRUS 811 * - r Downloaded from FIG. 6. Spider monkey foamy virus-infected HEK cells. Tubular membrane profiles engulfing nucleoids are seen to enclose a particle and form a complete virion. x 72, on July 7, 2018 by guest grivet (Cercopithecus aethiops), squirrel (Saimiri), African bushbaby (Galago), bonnet (Macaca radiata), and the chimpanzee (Pan) (7-9, 16, 19, 20). This is the first report of an apparently species-specific foamy virus isolated from a spider monkey (Ateles). This virus has not shown an antigenic relationship by neutralization to the seven serotypes of simian foamy viruses. In addition, antisera prepared against the two herpesviruses isolated from spider monkeys, herpes ateles and spider monkey herpesviruses, do not neutralize the spider monkey foamy virus (7, 12, 13). The spider monkey foamy virus displays the basic characteristics of syncytium-forming viruses of simian, bovine, and feline origin. This virus is not inhibited by IUdR and is associated with an RNA-dependent DNA polymerase, indicating that it is an RNA virus. In addition, the virus induces syncytia in vitro, replicates in

9 812 HOOKS ET AL. INFECT. IMMUNITY TABLE 3. Cross-neutralizing antibody to simian foamy viruses Reference antisera to:' Simian foamy virus Spider monkey Spider Simian foamy virus type type (S-140) sera monkey foamy virus Spider monkey foamy a Antisera were screened at a 1:10 dilution with approximately 100 TCD5o virus inocula. TABLE 4. Distribution of neutralizing antibody to simian foamy viruses in spider monkey sera Simian foamy virus type Spider monkey seraa at dilution of 1:4 1:10 1 0/13b 2 0/12 3 0/8 4 0/7 5 0/2 6 0/7 0/13 7 1/7 0/8 Spider monkey foamy 19/31 5/11 a Sera were considered positive if the sera neutralized 100 TCD5O virus inocula in HEK tissue culture. bnumber positive sera/number of sera tested. the cytoplasm, and is chloroform sensitive. The virus persists in the natural host in the presence of circulating antibody. Low concentrations of BUdR markedly reduced the spider monkey foamy virus infectivity. In contrast, IUdR did not significantly reduce the virus infectivity. The fact that IUdR does not significantly inhibit simian foamy virus production has been reported (6, 9, 21). Inhibition of simian foamy virus by BUdR and the lack of significant inhibition by IUdR was observed by Parks and Todaro (14). These authors suggested that since the brominated derivative of uridine resembles thymidine more closely than does the iodinated derivative, the foamy virus reverse transcriptase may be able to discriminate between the two halogenated thymidine analogues. By electron microscopy, the spider monkey isolate was observed only within the cytoplasm of infected cells. The time of collection of TABLE 5. Neutralizing antibody to spider monkey foamy virus in sera from man and other species Species Species Spider monkey foamy virus neutralizing antibodya at dilu- ~~~~~tionof 1:4 1:10 Human Animal caretakers... 0/7b Laboratory personnel.0/9 Other... 0/1 Spider monkey (Ateles) /31 Chimpanzee (Pan)... 0/10 Rhesus monkey (Macaca mulatta)... 0/9 Guinea pig... 0/6 0/2 Rabbit... 0/1 0/11 Hamster (pool)... 0/4 Goat..0/5 a Sera were considered positive if the sera neutralized 100 TCD5O virus inocula in HEK tissue culture. b Number positive sera/number of sera tested. infected cells and the method of fixation could account for the absence of both extracellular particles and particles with spikes. Extracellular particles with spikes have been observed with the other simian foamy viruses (3). The spider monkey foamy isolate closely resembles the bovine syncytial virus particles reported by Boothe et al. (2). Bovine syncytial virusinfected cells contained a morphological variation consisting of endoplasmic reticulumassociated (ERA) virus particles. The nucleocapsids of the ERA particles were 35 to 45 nm in diameter and were enveloped by a double membrane. The nucleocapsids observed in this study were 45 to 50 nm in diameter and were enveloped by a double membrane in a manner

10 VOL. 8, 1973 NEW SPIDER MONKEY FOAMY VIRUS 813 similar to the bovine syncytial virus ERA particles. Because of the presence of a reverse transcriptase and morphological similarities to the oncogenic RNA viruses, the syncytium-forming viruses have gained renewed interest as a possible member of the oncornavirus groups. Achong et al. (1) reported the isolation of a foamy virus from human tissue, but this has yet to be confirmed. Even though the bovine, feline, simian, and human syncytium-forming viruses have been isolated from malignant tissues, the relationship between the viruses and the abnormality of the tissue is questionable (1, 10, 11, 22). To date, these viruses have not induced tumors in experimental animals and have as yet to be assigned an etiological role in human and animal diseases. Efforts to induce clinical disease in experimental animals with the simian foamy viruses are in progress. LITERATURE CITED 1. Achong, B. G., P. W. A. Mansell, M. A. Epstein, and P. Clifford An unusual virus in cultures from a human nasopharyngeal carcinoma. J. Nat. Cancer Inst. 46: Boothe, A. D., M. J. Van Der Maaten, and W. A. Malmquist Morphological variation of a syncytial virus from lymphosarcomatous and apparently normal cattle. Arch. Gesamte Virusforsch. 31: Chopra, H. C., J. J. Hooks, M. J. Walling, and C. J. Gibbs, Jr Morphology of simian foamy viruses, with particular reference to virus isolated from spontaneous tumor of a rhesus monkey. J. Nat. Cancer Inst. 48: Feldman, H., and S. Wang Sensitivity of various viruses to chloroform. Proc. Soc. Exp. Biol. Med. 106: Gajdusek, D. C., N. G. Rogers, M. Basnight, C. J. Gibbs, Jr., and M. Alpers Transmission experiments with kuru in chimpanzees and the isolation of latent viruses from explanted tissues of affected animals. Ann. N.Y. Acad. Sci. 162: Hooks, J. J., C. J. Gibbs, Jr., E. C. Cutchins, N. G. Rogers, P. Lampert, and D. C. Gajdusek Characterization and distribution of two new foamy viruses isolated from chimpanzees. Arch. Gesamte Virusforsch. 38: Hull, R. N The simian viruses. Virol. Monogr., vol. 2. Springer-Verlag, New York. 8. Johnston, P Taxonomic features of seven serotypes of simian and ape foamy viruses. Infect. Immunity 3: Johnston, P A second immunologic type of simian foamy virus: monkey throat infections and unmasking by both types. J. Infect. Dis. 109: McKissick, G. E., and P. H. Lamont Characteristics of a virus isolated from a feline fibrosarcoma. J. Virol. 5: Malmquist, W. A., M. J. Van Der Maaten, and A. D. Boothe Isolation, immunodiffusion, immunofluorescence and electron microscopy of a syncytial virus of lymphosarcomatous and apparently normal cattle. Cancer Res. 29: Melendez, L. V., R. D. Hunt, M. D. Daniel, B. J. Blake, and F. G. Garcia Acute lymphocytic leukemia in owl monkeys inoculated with herpesvirus saimiri. Science 171: Melendez, L. V., R. D. Hunt, N. W. King, H. H. Barahona, M. D. Daniel, C. E. 0. Fraser, and F. G. Garcia Herpesvirus ateles, a new lymphoma virus of monkeys. Nature N. Biol. 235: Parks, W. P., and G. J. Todaro Biological properties of syncytium-forming ("foamy") viruses. Virology 47: Parks, W., G. Todaro, E. M. Scolnick, and S. Aaronson RNA dependent DNA polymerase in primate syncytium-forming (foamy) viruses. Nature (London) 229: Rogers, N. G., M. Basnight, C. J. Gibbs, Jr., and D. C. Gajdusek Latent viruses in chimpanzees with experimental kuru. Nature (London) 216: Rosen, L Hemagglutination with animal viruses, p In K. Habel and N. Salzman (ed.), Fundamental techniques in virology. Academic Press Inc., New York. 18. Ross, J., E. M. Scolnick, G. J. Todaro, and S. A. Aaronson Separation of murine cellular and murine leukaemia virus DNA polymerases. Nature N. Biol. 231: Rustigian, R., P. Johnston, and H. Reihart Infection of monkey kidney tissue cultures with virus-like agents. Proc. Soc. Exp. Biol. Med. 88: Scolnick, E. M., W. P. Parks, G. J. Todaro, and S. A. Aaronson Immunological characterization of primate C-type virus reverse transcriptase. Nature N. Biol. 235: Stiles, G. E., J. L. Bittle, and V. J. Cabasso Comparison of simian foamy virus strains including a new serological type. Nature (London) 201: Valerio, M., J. C. Landon, and J. R. M. Innes Neoplastic diseases in simians. J. Nat. Cancer Inst. 40: Vogel, J., and A. Shelokov Adsorption-hemagglutination test for influenza virus in monkey kidney tissue culture. Science 126: