Vol. 14, No. 1 Printed in U.S.A.

INFECTION AND IMMUNITY, JU -V 1976, p. 225-231 Copyright (© 1976 American Society for Microbiology

Identification of the Stumptailed Macaque Virus Papovavirus

as a

New

MAGDALENA REISSIG,* THOMAS J. KELLY, JR., RICHARD W. DANIEL, S. R. S. RANGAN, AND KEERTI V. SHAH

Department ofPathobiology,* School of Hygiene and Public Health, and Department of Microbiology, School of Medicine, The Johns Hopkins University, Baltimore, Maryland 21205, and Deltd Regional Primate Research Center, Tulane University, Covington, Louisiana, 70433 Received for publication 10 February 1976

A new viral agent, stumptailed macaque virus (STMV), isolated from uninoculated stumptailed macaque kidney cultures was identified. The virions had the size and morphology of papovaviruses of the simian virus 40 (SV40)-polyoma subgroup, but many of them appeared to have an additional outer envelope. The deoxyribonucleic acid of STMV was a superhelical circular molecule, with a mean length 91% of that of SV40. The antigenic relationship of this virus with other members of the group was examined by immune electron microscopy of isolated virions and by immunofluorescent staining of virus-infected cells. STMV was immunologically distinct from SV40, BK virus (BKV), polyoma virus, and JC virus. Its tumor antigen may be related to those of SV40 and BKV.

Rangan et al. (16) reported that uninoculated cultures from kidneys of the stumptailed macaque (Macaca speciosa) developed cytoplasmic vacuolation after a few passages, and that many cells in these vacuolated cultures contained both intranuclear and cytoplasmic virus particles. This stumptailed macaque virus (STMV) was tentatively identified as a new papovavirus of the simian virus 40 (SV40)-polyoma subgroup, on the basis of virion morphology and immunofluorescent (IF) reactivity of STMV-infected stumptailed monkey kidney (STK) cultures (K. V. Shah, S. R. S. Rangan, M. Reissig, and J. Roberts, Fed. Proc. 34:4278, 1974). STMV does not produce a cytopathic effect in a wide variety of cell cultures (16), but it has now been adapted to grow in primary rhesus kidney (RK) cultures (Shah, unpublished data). This report describes the identification of STMV as a new, immunologically distinct papovavirus. The size and structure of STMV deoxyribonucleic acid (DNA) were determined, and the antigenic relationship of STMV with other members of the SV40polyoma subgroup was studied by immune electron microscopy (EEM) of isolated virions and by IF tests of virus-infected cells. MATERIALS AND METHODS STMV preparations. STMV derived from the kidneys of stumptailed macaque Delta no. 3919 and grown in primary RK cells (virus stock GM-RK1) was concentrated and used for DNA extraction, IEM, and preparation of STMV-immune rabbit antisera. Virions with identical characteristics were

also isolated from vacuolated kidney cultures derived from five fetal and six adult stumptailed macaques (K. V. Shah, R. W. Daniel, M. Reissig, S. R. S. Rangan, and F. Z. Beluhan, Fed. Proc. 35:2013, 1976). The infected cultures with their medium were frozen and thawed on a dry-ice-alcohol mixture to disrupt the cells, and the suspension was clarified by centrifugation at 10,000 x g for 1 h. The supernatant virus was concentrated 20- to 50-fold by centrifugation at 100,000 x g for 1 h. Purification and characterization of STMV DNA. The crude virus preparation obtained as described above was made 0.6% in sodium dodecyl sulfate (SDS) and 1 mg/ml in Pronase and incubated at 37 C for 5 h. The resulting solution was extracted twice with phenol saturated with 0.1 M tris(hydroxymethyl)aminomethane-hydrochloride, pH 8.6, and dialyzed against TE [0.01 M tris(hydroxymethyl)aminomethane-hydrochloride, pH 8.6, 0.001 M ethylenediaminetetraacetate]. The viral DNA was separated from contaminating host DNA by centrifugation to equilibrium in CsCl (p = 1.56) containing 200 ,.Lg of ethidium bromide per ml (15). The band of covalently closed molecules (form I) was collected, passed over a Dowex-50 column to remove ethidium bromide, and dialyzed against TE. Electron microscopy of DNA was carried out as described by Davis et al. (2). Contour length measurements were made using a Numonics digital length calculator. Immune sera. STMV-immune sera were prepared in juvenile rhesus free from SV40 antibodies and in rabbits. Each of two rhesus was given a single intravenous (i.v.) injection of 0.4 ml of concentrated STMV obtained from the fifth to seventh passage of vacuolated kidney cultures from stumptailed macaque no. 3919. The inoculated monkeys were bled out 3 to 4 weeks after inoculation. Each of two 225

226

REISSIG ET AL.

INFECT. IMMUN.

rabbits received two intravenous injections of con- distilled water and two drops of a 3.2% solution of centrated RK-adapted STMV 1 week apart and potassium phosphotungstate, pH 7.0, containing a was bled out 10 days after the second injection. All of known number of latex spheres (23) 109 nm in diamthe inocula contained at least 108-0 STMV virions as eter. A drop of the mixture was placed with a capilestimated by electron microscopic examination after lary pipette on 500-mesh Formvar-carbon-coated negative staining of virus preparations containing a electron microscope grids, and excess fluid was reknown number of latex spheres (23). The hyperimmune sera against SV40, BK virus (BKV) (5), and JC virus (JCV) (12) were prepared by six weekly .'t intravenous injections of the respective viruses into rabbits (13) and were provided by 0. Narayan. The SV40-immune rhesus serum was collected 3 weeks after inoculation of about 105°0 mean tissue culture infective doses of SV40. An anti-SV40 rabbit serum prepared against SDS-disrupted (anti-SDS) empty SV40 virions (11) was kindly provided by H. Ozer. The antipolyoma serum was prepared by three weekly intraperitoneal inoculations of live virus into Swiss mice and was kindly supplied by the Office of Program Resources and Logistics, Viral Oncology, National Cancer Institute. Preparation of virions for IEM. In crude preparations of STMV, a considerable proportion of the virions appeared to be enveloped, and non-enveloped virions were often aggregated. Therefore, for IEM studies, the pellets containing STMV virions were treated with Genetron-113 (7) for 10 min to dissociate the virions and remove cell membranes and viral envelopes. BKV, SV40, and polyoma virions were obtained by infection, respectively of W138 cells, primary African green monkey kidney cells, and primary mouse embryo cells and were concentrated by centrifugation at 100,000 x g for 1 h. These virions did not require treatment with Genetron113. JCV prepared by Genetron treatment of the brain of a patient with progressive multifocal leukoencephalopathy (14) was kindly supplied by 0. Narayan. IEM. Three drops of the virus suspension was FIG. 1. Negatively stained preparation of unmixed with three drops of the antiserum dilution in treated STMV virions from an uninoculated, fetal distilled water and incubated for 1 h at 37 C. The STK culture. The naked virions appear partially mixture was centrifuged at 100,000 x g for 1 h. The coated by amorphous material. Two enveloped viriwell-drained pellet was resuspended in one drop of ons are present. Magnification: x220,000. * i'

TABLE 1. Reactivity of STMV virions and anti-STMV sera in immune electron microscopy No. of virions coated/no. of virions examined

Antiserum and dilution

_

_ Polyoma

_

_ Jcv

Virionsa _ BKV

Polyoma, mouse, 1:5 123/123 JC virus, rabbit, 1:5 31/72 BK virus, rabbit, 1:5 111/116 SV40, rhesus, 1:5 0/52* SV40, rabbit, 1:5 SV40 (anti-SDS), rabbit, 1:20 0/53 0/55 123/127 SV40 (anti-SDS), rabbit, 1:100 0/61* STMV, rhesus, 1:5 0/53 0/51 STMV, rhesus, 1:5 0/52 0/51 STMV, rabbit, 1:5 0/56 0/52 0/53 Normal rabbit, 1:5 ( Positive specimens are italicized. Asterisks indicate that at least one-third were in aggregates of three virions or more.

__ SV40

0/115* 61/66 43/60 54/59 90/101

_ STMV

0/60* 0/58 3/56* 0/54 0/58* 0/52*

26/51 29/63 57/63 0/55 of the noncoated particles 0/50 0/51 0/57

VOL. 14, 1976

STUMPTAILED MACAQUE PAPOVAVIRUS

moved with the same pipette until only a thin layer of fluid remained. The grids were examined with a Siemens Elmiskop I electron microscope at 80 kV at magnifications ranging from x 10,000 to x 40,000. At least two grids were examined for each virus-antiserum mixture, and a minimum of 50 virions were scored for antibody coating and for aggregation (8). A sample was considered positive when one-third or more of the virions appeared to be coated by antibody. Specimens that were negative for antibody coating, but in which noncoated particles forming aggregates of three or more virions comprised at least one-third of the total number of virions, are identified in Table 1 by asterisks. All tests were done on coded samples. IF tests. Antigen preparations were made on LabTek (Miles Laboratory, Naperville, Ill.) chamber slides. STMV antigen was prepared either by inoculation of primary RK cells with rhesus-adapted STMV, or by harvesting vacuolated STK cultures from stumptailed macaque Delta no. 3919. W138, AGMK, and 3T3/D mouse cells inoculated, respectively, with BKV, SV40, and polyoma virus were harvested at 3 to 4 days postinoculation to obtain cells acutely infected with these viruses. Cells from an SV40-transformed hamster line were used as a source for SV40 tumor (T) antigen. BKV T antigen was obtained from the CS cell line (19), derived from a BKV-induced tumor. The lowest serum dilutions tested were undiluted or 1:5. All tests were done by

227

the indirect method as previously described (17) using fluorescein-labeled antibodies against human, rabbit, hamster, and mouse immunoglobulins (Meloy Laboratories, Springfield, Va.). Hemagglutination-inhibition (HI) and neutralization tests. The STMV-immune sera were tested for their ability to inhibit polyoma (3) and BKV hemagglutination (20) and to neutralize SV40 virus (17).

RESULTS Morphology of the virions. Negatively stained preparations of untreated virus from all 11 virus preparations contained naked as well as apparently enveloped virions. The naked virions measured 40 to 45 nm in diameter; they resembled papovaviruses of the SV40-polyoma subgroup, except that they appeared irregularly and incompletely coated by a fine amorphous material. Such particles were often aggregated, and their capsomers were not clearly outlined. The enveloped virions measured 55 to 60 nm, and their capsid was identical to that of the naked virions (Fig. 1 and 2). The nature and origin of these apparent envelopes is not known. Enveloped particles were observed only infrequently in preparations of the other papovaviruses. Genetron treatment resulted in the

FIG. 2. Negatively stained preparation of untreated STMV virions from a rhesus kidney culture inoculated with STMV. The virions are apparently enveloped. Magnification: x150,000.

228

INFECT. IMMUN.

REISSIG ET AL.

disaggregation of the STMV virions and in the removal of the viral envelopes as well as of most of the amorphous coating around the particles. Structure and molecular weight of STMV DNA. Preparations of STMV yielded covalently closed, duplex DNA molecules (form I), which were easily purified by methods used previously for the purification of the DNAs of other papovaviruses (see Materials and Methods). The final preparations contained over 99% circular molecules as assayed by electron microscopy. The contour length of form I STMV DNA was measured against linear SV40 DNA molecules (prepared using R.EcoRI endonuclease [9, 10]) present on the same grid (Fig. 3). The STMV molecules formed a homogeneous population with a mean length 0.910 + 0.006 (standard error) of that of SV40. This corresponds to a molecular weight of approximately 3.0 x 106, taking the molecular weight of SV40 DNA as 3.28 x 106 (6, 22). Antigenic relationships of STMV. In IEM

tests, virus-antibody qomplexes were observed as a filamentous network, completely covering the surface of the naked virions (Fig. 4); coated virions occurred only in the presence of specific antiserum. Although clusters of coated virions were often seen, virion aggregation was not characteristic of virus-antiserum interaction. Since it has also been reported by Kapikian et al. that particles heavily coated with antibody often fail to form aggregates (8), the scoring of a sample as positive was based on antibody coating and not on aggregation. The results are summarized on Table 1. All the virion preparations reacted satisfactorily with the homologous sera; in every case at least 40% of the virions appeared coated by antibody and, except in the case of JCV virions, 90% or more of the virions were coated by at least one of the homologous antisera. There was no evidence of cross-reactivity between STMV and the other papovaviruses as judged by antibody coating. STMV virions were not coated by antibodies to SV40 (Fig. 5), BKV, JCV, or polyoma

I .

.

%.,.

.j.

1

Identification of the stumptailed macaque virus as a new papovavirus.

Vol. 14, No. 1 Printed in U.S.A. INFECTION AND IMMUNITY, JU -V 1976, p. 225-231 Copyright (© 1976 American Society for Microbiology Identification o...
2MB Sizes 0 Downloads 0 Views