JOURNAL OF VIROLOGY, Apr. 1992, p. 2143-21490022-538X/92/042143-07$02.00/0Copyright C 1992, American Society for Microbiology

Quantitation of a Lentivirus in Its Natural Host: SimianImmunodeficiency Virus in African Green Monkeys

SUSANNE HARTUNG, KLAUS BOLLER, KLAUS CICHUTEK, STEPHEN G. NORLEY,AND REINHARD KURTH*

Paul-Ehrlich-Institut, Paul-Ehrlich-Strasse 51-59, 6070 Langen 1, Germany

Received 20 August 1991/Accepted 20 December 1991

We have examined the viral load in the peripheral blood of simian immunodeficiency virus (SIV)-infectedAfrican green monkeys with a view to the unexplained apathogenicity of African green monkey SIV (SIVagm)in its natural host. By using polymerase chain reaction, viral DNA was detected in fresh peripheral bloodmononuclear cells (PBMC) of each of nine seropositive animals. The virus DNA load was variable among themonkeys tested, ranging from 5 to 50 (mean = 15) copies per 105 PBMC, which is comparable to that of humanimmunodeficiency virus type 1 (HIV-1) in humans. The level of infectious SIVagm in PBMC was measured byendpoint dilution cultures. SIVW. was recovered from PBMC from 14 of 17 antibody-positive monkeys (82%),and the mean SIVga titer in PBMC of seropositive African green monkeys was 10 tissue culture infectiousdoses per 106 cells, similar to the titer shown for HIV in asymptomatic carriers. Free infectious virus wasisolated from the plasma of 4 of 17 monkeys (24%), and SIVag expression in peripheral blood in vivo, asdemonstrated by in situ hybridization, was detectable only in those animals which were viremic. SIVgmreplication is therefore not totally suppressed in vivo, and SIVga has a viral load equivalent to that seen forHIV-1 in asymptomatic humans.

Simian immunodeficiency viruses (SIV) have been iso-lated from a variety of different primate species. Theyrepresent the closest known relatives of human immunode-ficiency virus (HIV) on the basis of their genetic, biological,and antigenic similarities. Therefore, these viruses are suit-able as a model for studying the pathogenesis of HIVinfection as well as performing extensive vaccination andchallenge experiments (18). SIV isolated from African greenmonkeys (AGMs) (SIVagm) (5, 10, 12, 17, 23) has twoproperties which make it particularly interesting: first, theAGMs represent the natural host of the virus, as demon-strated by the presence of virus-specific antibodies in a largeproportion of wild animals (15), and second, SIVagm inducesno disease in its natural host. One possible reason for thefailure of SIVagm to induce an AIDS-like disease could be thenature or strength of the immune response in infectedAGMs. However, in a recent report we showed that thefunctional immune response of AGMs to SIVagm is verysimilar to the response of humans to HIV (22). Moreover, wefind that the degree of sequence variation in vivo followinginfection of AGMs with molecularly cloned virus is compa-rable to that reported for HIV (2). Another possible expla-nation of why SIVag-infected AGMs remain healthy couldbe that infection of T cells in vivo does not result in aproductive virus infection. For HIV type 1 (HIV-1), thefinding of infectious virus in the plasma of both symptomaticand asymptomatic patients (7, 9, 13) shows that replicationin the host is not completely suppressed.

In this article, we report that the viral load in the periph-eral blood of infected AGMs is comparable to that seen inthe blood of HIV-1-infected patients. By using polymerasechain reaction (PCR), SIVagn, sequences were detected in100% of DNA samples from seropositive AGMs (5 to 50copies per 105 peripheral blood mononuclear cells [PBMC]),and we found that in infected monkeys, approximately 1 in

* Corresponding author.

100,000 PBMC produced infectious SIV. m. The detection ofviremia in 4 of the 17 plasma sampfes tested and thedemonstration of viral mRNA expression in unstimulatedPBMC from the viremic animals indicate that AGMs un-dergo infection with an actively replicating lentivirus.

MATERIALS AND METHODS

Animals. Blood was obtained from 21 adult AGMs (8 to 12years old) which had been imported from Ethiopia andKenya during the years 1978 to 1982 to the primate colony atthe Paul Ehrlich Institute. Of these animals, 16 displayed along-term (>6-year) natural infection by SIVagm, as judgedby their prolonged seropositivity; one seroconverted in 1988,and four seronegative AGMs served as controls. All animalswere and remain clinically healthy.

Cells and cell preparation. Molt 4 clone 8 (Molt 4/8) cells(16) were kindly donated by M. Hayami, Kyoto University.Molt 4/8-SAM is a chronically SIVagm-infected line estab-lished in our laboratory. PBMC were obtained by Ficoll-Hypaque density gradient centrifugation of whole blood.The PBMC layer was removed and washed twice in phos-phate-buffered saline (PBS). Cells for use in PCR studieswere pelleted and stored at -70°C until use.PBMC cultures. Decreasing numbers of PBMC (106; 105; 5

x 104; 104; 103) were stimulated with 200 ±g of phytohemag-glutinin (PHA) per ml and cocultivated directly with 2 x 106Molt 4/8 cells in RPMI 1640 with 20% fetal calf serum (FCS)and antibiotics. Coculture supernatants were subsequentlymonitored weekly for the presence of reverse transcriptase(RT) activity for 8 weeks. During this period, the mediumwas replaced by fresh medium (RPMI 1640 with 10% FCSand antibiotics) every 2 to 3 days. The lowest number ofPBMC which resulted in a positive culture was taken as theendpoint, and the titers of infectious SIVagm were thenexpressed as tissue culture infectious doses (TCID) per 106PBMC.

In parallel, 107 PBMC were stimulated with 200 ,ug ofPHA

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per ml for 24 h and cultured in medium (RPMI 1640 with 20%FCS and antibiotics) containing 10% interleukin-2 (BiotestGmbH, Dreieich, Germany). After 4 days, these cells werecocultivated with Molt 4/8 cells.

Plasma culture. After Ficoll-Hypaque centrifugation, theplasma, diluted 1:2 with PBS, was removed and centrifugedat 3,000 x g for 20 min. Subsequently, 10, 1, 0.1, and 0.01 mlof plasma, containing 10 IU of heparin per ml, were eachadded to 2 x 106 Molt 4/8 cells. Following incubation for 1 hat 37°C, cells were centrifuged to remove the plasma andmaintained in culture for up to 8 weeks. During this period,cultures were monitored weekly for the presence of RTactivity and medium was replenished every 2 to 3 days. Theplasma SIVagm titer was expressed as the number of TCIDper milliliter of plasma.To test the sensitivity of the isolation procedure and to

provide an internal control, 10 and 1 50% TCID (TCID501from a precisely titrated virus stock (22) of SIVagm (10TCID50/ml) were each added to 10 ml of plasma from aseronegative AGM containing 10 IU of heparin per ml (fourreplicates per dilution). After incubation of the plasma with2 x 106 Molt 4/8 cells for 1 h at 37°C, cells were centrifugedand cultured as described above.ELISA. Sera, diluted 1:100 to 1:12,800, were assayed for

the presence of antibodies able to bind viral antigen (purifieddisrupted SIVagm) by using standard enzyme-linked im-munosorbent assay (ELISA) protocols (19). Briefly, sucrosegradient-purified SIVagm, disrupted in 1% Nonidet P-40 anddiluted in water to 20 ,ug/ml, was dried overnight ontoflexible microtiter plates (50 ,ul per well). Plates were washedand incubated with blocking buffer (PBS, 3% bovine serumalbumin, 10% FCS) before further washing and addition ofsera diluted in blocking buffer plus 0.1% Tween 20. Afterincubation for 1 h at 37°C, the plates were washed andincubated with goat anti-human immunoglobulin G peroxi-dase conjugate (Sigma) diluted 1:500. Finally plates werewashed and incubated with substrate buffer (1 mg of o-phe-nylenediamine per ml of PBS [pH 6] plus 0.025% H202)before the reaction was stopped with 1 M H2SO4 and opticaldensity at 492 nm (OD492) was measured. Titers were definedas serum dilutions giving an OD that was 50% of themaximum plateau OD.RT assay. RT was assayed as described previously (28).Oligonucleotides and radioactive probes. For amplification

of SIVagm sequences, the following primer pairs were used:(i) SAG-gag220+ (5' TGG CGC CCG AAC AGG GAC(T/C)TG 3') and SAGgag729- (5' GCT TC(C/T) TCT GTGTCT TTC ACT T 3') and (ii) SAGpoI2947+ (5' CCA CCTTAT GAG TGG ATG GGA T 3') and SAGpol4207- (5' CTTCTA TGA ATC CAC TGG CTA C 3'). For detection of theamplified products, a full-length EcoRI insert of clonedSIVagm3 (4) 32p labeled by nick translation was appliedduring Southern hybridization. For in situ hybridization, along terminal repeat-derived 35S-labeled RNA probe wasprepared by in vitro transcription of an antisense-strandEcoRI-XhoI insert of clone SIVagm3 in the Bluescript plas-mid (3) and alkali hydrolyzed as described previously (8).The 2.5-kbp-long fragment spanned the 3' part of the ge-nome, including one complete long terminal repeat.PCR. DNA from 107 PBMC was prepared according to

standard methods involving lysis of the cells in the presenceof sodium dodecyl sulfate, digestion with proteinase K at56°C for 12 h, and phenol, phenol-chloroform, and chloro-form extractions. After ethanol precipitation in the presenceof 0.3 M sodium acetate and centrifugation, the DNA waswashed with 70% ethanol, redissolved in water, and quanti-

fied by measuring OD260. The DNA was melted at 94°C for10 min and rapidly cooled to 15°C. The PCR was performedas described previously (25), with slight modifications.Briefly, DNA equivalent to that of 105 cells (approximately 1,ug of genomic DNA) was added to the PCR mixture con-taining S puM each primer, 200 puM each deoxynucleosidetriphosphate, 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mMMgCl2, 0.01% gelatin, and 2.5 U of Taq polymerase. Subse-quently, 30 to 35 cycles of denaturation (94°C; 45 s), hybrid-ization (45°C; 1.5 min), and extension (72°C; 1.5 min) wereperformed. Complete reactions were separated by size byusing 1.2% agarose electrophoresis, and the subsequentSouthern analysis was performed by using standard proce-dures (26). For the standard curve, dilutions of clonedSIVagm3 DNA (in linearized plasmid form) were mixed with1 pug of carrier DNA from Molt 4/8 cells and analyzed asdescribed above.

In situ hybridization. Cells were fixed with 4% formalde-hyde in PBS, dried on Silan-coated slides (24), and dehy-drated in a graded series of ethanol. Cell pretreatment and insitu hybridization were performed according to the methodof Cox et al. (8). The 3 S-labeled RNA probe (prepared asdescribed above) was diluted to about 1 ptg/ml in hybridiza-tion buffer containing 50% formamide, 0.6 M NaCl, lxDenhardt's solution, 10 mM Tris-HCl (pH 7.5), 1 mMEDTA, 0.5 mg of tRNA per ml, and 100 pug of poly(A) RNAper ml. Hybridization was performed overnight at 55°C. Theslides were then rinsed for 24 h at 55°C in 50% formamide-10mM Tris-HCI (pH 7.5)-i mM EDTA-0.6 M NaCl. Controlswere prepared by the same procedure with the sense strandof our probe or by pretreating the slides with 40 ,ug of RNaseA per ml for 20 min prior to hybridization. For autoradio-graphic detection of hybridized RNA, the sections werecovered with Kodak nuclear emulsion NTB-2. After expo-sure for 2 to 14 days, the slides were developed in Dectol(Kodak), fixed, stained with hematoxylin-eosin, and exam-ined with a Zeiss Axiophot microscope.

RESULTS

Determination of SIV2gm DNA copy number. To quantitatethe number of SIVagm DNA copies present in the PBMC ofinfected AGMs, PBMC lysates from nine seropositive mon-keys were subjected to PCR. By using two different oligo-nucleotide primer pairs specific for the gag and pol genes ofall known molecular clones of SIVagm (3, 11, 14), viralsequences were detected in the DNA of all nine animals.Southern analysis of the amplificates with molecular virusclone SIVagm3 as the radioactive probe resulted in specificbands of 0.5 and 1.2 kbp for the gag and pol genes,respectively. In order to estimate the number of viral DNAcopies present in PBMC of infected monkeys, the signalintensities from all amplifications were compared by densi-tometer scanning with the signal intensity resulting fromamplification of 101 to 104 copies of a molecularly clonedSIVa m3 DNA. Because of variability of the pol-specificsignal (resulting presumably from differences in the degree ofhomology between the various viruses and the primersused), the gag-specific signal was used throughout for quan-tification. As shown in Fig. 1, as few as 10 copies of standardviral DNA were detected, and there was a linear relationshipbetween the signal intensity and the amount of viral DNApresent in the reaction. In the analysis of the PBMC, thenumber of SIVagm DNA copies ranged from 5 (by extrapo-lation of the standard curve) to 50 per 105 cells in differentmonkeys (Table 1).

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SIVagm VIRUS LOAD 2145

6 7 8

pol

Z gag

10 - lt)3 104# SlVagry3 copics

FIG. 1. Quantitation of viral DNA of SIVagm- (a) 0, 10, 102, 103,and 104 (lanes 0 to 4) copies of molecular virus clone SIVagm3 wereused in single PCR reactions with the gag and pol primer pairsdescribed in Materials and Methods. Lanes 5 to 8, signals from PCRreactions with PBMC DNA (approximately 1 ,ug of genomic DNA)of one seronegative AGM (AGM 5, signal intensity = 0 [lane 5]) andthree seropositive AGMs (AGM 2a, signal intensity = 1.7 [lane 6];AGM 4, signal intensity = 2.1 [lane 7]; AGM 43, signal intensity =2.6 [lane 8]). (b) A linear correlation of gag- and pol-specific signalintensities with the viral DNA copy number was observed within therange of 10 to 104 molecules of cloned SIVagm3. The gag-specificsignal was used for quantitation because of the higher variability ofpol among different SIVag, strains (3). *, derived from densitometerreadout and expressed as arbitrary units.

Isolation of SIVgm in PBMC. To measure the level ofinfectious SIVagm present in AGM PBMC, we performedcocultivation assays with decreasing numbers of PHA-stim-ulated PBMC (106; 10(; 5 x 104; 104; 103). SIVagm wasisolated from 14 of 17 (82%) antibody-positive AGMs. Fromthree animals (AGMs 8, 15, and 33), virus could not berescued (in each of three attempts), even when 107 PBMCstimulated for 4 days were cocultivated with Molt 4/8 cells.All four seronegative animals yielded negative cultures. TheSIVagm titers in the 17 seropositive monkeys ranged from 0to 20 (mean = 10) TCID/106 cells (Table 1). Repeat titrationsof PBMC from six of the culture-positive monkeys (AGMs2a, 24, 43, Z2, Z8, and Z14) performed 3 to 6 months afterthe initial titrations yielded titers the same as or within 1

TABLE 1. In vivo virus load of SIVagm

Animal TCID/106 TCID/ml Viral DNA No. of RNA- Serumno. PBMCI of plasmab (no. of copies! expressing cells/ titerei0-1 cells)C 106 PBMCd

5 0 0 0 0 <10013 0 0 < 10032 0 0 - 0 < 100

8 0 0 0 2,81815 0 0 - - 3,16233 0 0 0 2,8182a 1 20 5 4 4,4674 1 0 10 0 1,58526 1 0 0 3,548Z2 1 0 - 2,239Z8 1 0 10 - 1,58516 10 0 5 0 7,07924 10 0 7,94343 10 2 50 4 5,012Z4 10 0 0 3,162Z13 10 0 5 3,981Z14 10 0 - 3,5483 20 0 - 0 2,23937 20 2 40 1 3,54838 20 0.2 5 2,818

Determined by culture of serially diluted AGM PBMC in Molt 4/8 cells.b Determined by culture of serially diluted AGM plasma in Molt 4/8 cells.c Estimated from the intensity of the gag-specific signal in PCR (Fig. 1).d Estimated by in situ hybridization of unstimulated AGM PBMC.e Measured by standard ELISA of AGM sera with whole SIVagm lysate as

the antigen. Results are expressed as the reciprocal dilution giving 50%maximal reaction.f-, not tested.

dilution step of the original titer. The levels of PBMCinfection in vivo therefore appeared to remain relativelystable.

Isolation of SIVagm from plasma. Plasma samples from 17seropositive and 4 seronegative animals were tested forcell-free, infectious SIVagm. Virus was isolated from 4 of 17(24%) antibody-positive monkeys but from none of theantibody-negative animals. By using serial 10-fold dilutions,it was shown that titers ranged from 0.2 to 20 TCID/ml(Table 1). Plasma samples from each of the viremic animals(AGMs 2a, 43, 37, and 38) were tested two to four times overa period of 6 months. Titers fluctuated but remained in therange stated above, except in the case of one animal (AGM38), which went from having a titer of 2 TCID/ml to beingaviremic within this period. Repeated attempts over a periodof 6 months to isolate virus from the plasma of four of theinitially aviremic animals (AGMs Z4, 8, 3, and 16) consis-tently failed. Since large volumes of plasma had to beincubated with Molt 4/8 cells, coagulation could be avoidedonly when heparin (10 IU/ml) was added to the plasma.Heparin is thought to inhibit HIV replication (1, 20), and totest the sensitivity of the isolation procedure used here andto provide an internal control, we spiked heparinized plasmaof a seronegative AGM with infectious SIVagm and at-tempted reisolation. Addition of 1 TCID50 to the plasmaresulted in a clearly positive signal in two of four plasmacultures, indicating that very low levels of infectious viruscould be isolated from heparinized plasma. All culturesreceiving plasma spiked with 10 TCID50 became positive.

In situ hybridization. By using in situ hybridization, weexamined SIVagm RNA expression in uncultured PBMC ofAGMs. Specificity of labeling was tested by using a prepa-ration of uninfected Molt 4/8 cells spiked with 10% Molt

a 0 1 2 3 4 5

_ *db

b

SignalIntcnsitv*

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2146 HARTUNG ET AL.

p~~~~~~~~.

a

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*~~~~~~~~~~~~~~~~~~~~4

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*5 -~~~~~~~~~ -~~~~n

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exposurwasfr3 das. (a)PBMC fom a sroposiive, vremia-ositiv AGM. b) PBM from he sam anima after7 daysof stiulatioin~vitrowitPH an nelui- ro ohbidzto.Mgiiain20

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SIVagm VIRUS LOAD 2147

4/8-SAM cells (chronically infected with SIVagm). As ex-pected, a strong labeling of 10% of the cells was observedwhen they were hybridized with the SIVagm-specific probes.In contrast, no labeling was found after RNase pretreatmentof Molt 4/8-SAM cells or by hybridization of Molt 4/8-SAMcells with the sense strand of the specific probe (data notshown).

Fresh PBMC (2.5 x 106) of 10 seropositive and 3 seroneg-ative AGMs were analyzed for expression of SIVagm RNA.PBMC from seven of the seropositive monkeys were con-sistently negative (as were those from the three seronegativemonkeys), even when autoradiographic exposure was pro-longed from 2 to 14 days. It had been possible to isolate virusfrom the PBMC but not from the plasma of six of theseanimals. PBMC from three of the monkeys showed a posi-tive hybridization signal in 1 to 4 of 106 cells (Fig. 2a). Thesewere the same animals which had been shown to be viremicby reisolation of SIVagm from plasma. In contrast, whenPBMC from all virus culture-positive monkeys were stimu-lated with PHA and cultured in medium containing interleu-kin-2 for 7 days prior to hybridization, SIVa m-expressingcells were detected at a frequency of up to 5%o (Fig. 2b).

DISCUSSION

The aim of this study was to compare the viral load inSIVagm-infected AGMs with the viral titer seen in HIV-infected humans, with a view to the unexplained apathoge-nicity of SIVagm in its natural host. HIV-1 infection inhumans results in a productive infection with a rise in virustiter during the progression of the disease (7, 13). If SIVagm-infected AGMs are able to control virus replication and keepthe infection in a latent state, this may be a reason for thelack of disease in infected animals.

Recent studies using PCR have estimated levels of HIVDNA in infected humans (healthy or with AIDS, Centers forDisease Control [CDC] class II to class IVc) to range from1.2 to 136 copies (mean = 12.5) per 105 cells (27). It was alsoestimated (6) that there are 5.3 to 83 (median = 9.1) HIV-1DNA copies per 105 CD4+ T cells in asymptomatic patients(CDC class II). When the PBMC of nine seropositive AGMswere subjected to PCR, viral sequences were detected ineach animal. By comparing PCR results obtained with thePBMC and those obtained with a standard SIVagm provirusclone, a quantitative estimation of the proviral load waspossible. With 5 to 50 copies (mean = 15) per 105 cells, theestimated viral load was similar to that shown for HIV-1-infected humans. It is not possible, however, to exactlycompare HIV-1 and SIVagm viral loads, as a wide range isobserved from one patient to another, giving varying meanvalues in the different published studies.

SIVagm DNA levels do not necessarily reflect the level ofinfectious virus in the monkeys' lymphocytes. Using end-point dilution cultures, we found that in seropositive AGMs,1 in 105 PBMC harbored infectious virus. The discrepancybetween SIV DNA levels and viral titers in PBMC couldtherefore be Mue to defective viral DNA, although it mightalso be caused by differences in the relative sensitivities ofthe two detection systems. The presence of large amounts ofdefective viral DNA copies in SIVagm-infected AGMs wasrecently shown by molecular cloning and partial sequencing(2). It has been suggested (21) that accumulation of defectiveproviruses is a critical factor in the development of AIDS.The fact that defective SIVagm DNA accumulates in AGMs,while in asymptomatic humans more than half of the copiesin CD4+ T cells were found to be replication competent (6),

indicates that other or additional factors are required fordisease development. Interestingly, AGMs with titers ofinfectious SIVa m of 10 and 20 TCID/106 PBMC were shownto have a signiffcantly higher serological response (0.01 <Pc 0.025) than animals with titers of < 1 TCID/106 PBMC(data not shown). This may indicate that periods of viremiaand the associated increase in the levels of viral antigenresult in a restimulation of the immune response. It isknown, however, that the titers of antibody as measured byELISA remain remarkably stable over periods of 4 to 5 yearsin these animals (data not shown), suggesting either thatperiods of viremia are scarce or that there is very littleimpact on antibody levels by changes in viral load.Asymptomatic patients infected with HIV-1 have approx-

imately 1 cell per 50,000 PBMC infected with a replication-competent virus (13) and a mean viral titer twofold higherthan that shown here for SIVagm. However, it is known thatAGM lymphocytes are only 15 to 20% CD4+ (lOa), less thanhalf of the subpopulation seen in humans. If viral loads arenormalized to the percentage of infected CD4+ cells in thePBMC, SIVagm would appear to have a replicative capacityin monkeys similar to or higher than that of HIV-1 inasymptomatic humans. In these experiments, total PBMCDNA was used for the PCR, and it is therefore not possibleto make any comments about the in vivo compartmentaliza-tion of infection. Ongoing experiments using cells sortedwith a fluorescence-activated cell sorter and then analyzedby PCR will give information concerning the precise celltypes infected in vivo, including the possibly crucial roleplayed by macrophages as reservoirs of virus.

Since the PBMC that were tested for the release of viruswere activated in culture, the observed viral load in PBMCof both AGMs and humans may not necessarily reflect thelevels of viral replication in vivo. By using in situ hybridiza-tion, it was possible to detect SIVagm RNA-expressing cells(in 2.5 x 106 freshly sampled PBMC) in only three animalswhich had been shown to be viremic, although PBMC fromall seropositive animals tested showed a positive signal afterPHA stimulation. In the nonviremic animals, the negativeresults with unstimulated cells strongly suggest that cellsexpressing SIVa mare very rare, constituting less than 1 in2.5 x 106 PBMC. In contrast, the viremic animals had 1 in0.25 x 106 to 1 x 106 cells expressing virus. A comparison ofthe results from in situ hybridization and endpoint PBMCdilution assays indicates a difference in viral expressionbetween viremic and nonviremic animals. Whereas nonvire-mic AGMs harbor the virus latently in at least 95% of theinfected cells, those which are viremic express viral RNA inup to 50% of the cells carrying replication-competent vi-ruses. In comparison, the proportion of latently infectednonproducing cells in patients with AIDS has been estimatedto be 99.6% of the infected lymphocytes (13).The frequency of plasma viremia among seropositive

AGMs was 24%, corresponding to the frequency found forHIV-1 patients of CDC class 11 (7), although the mean viralplasma titer of AGMs was fourfold lower. In contrast, in asecond study HIV-1 was detected in the plasma of everyseropositive patient, including 16 asymptomatic patients(13). This difference may reflect population variation, asdifferent classification systems were used. Interestingly, wewere not able to detect p28 antigen in the plasma of 40naturally infected AGMs, including the 4 viremic monkeys(data not shown). Because of the failure of AGMs to developantibodies against p28 (22), this observation cannot beexplained by trapping of p28 in antigen-antibody complexes.On the other hand, we used a commercial HIV-1 antigen

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capture assay (Abbott Laboratories), which is less sensitivefor SIVagm p28 than for HIV-1 p24, and these data maytherefore indicate levels of p28 below the detection limit (100pg/ml for highly purified SIVagm p28).

In summary, the viral DNA load of SIVagm-infectedAGMs is comparable to that of HIV-1 in infected patients.The levels of infectious virus in PBMC of infected AGMs aresomewhat lower than but comparable to the titers demon-strated for asymptomatic HIV-1 carriers. In accordance withthe findings obtained with the human-HIV system, very fewSIVagm-infected PBMC express the virus in vivo. The fre-quency of plasma viremia was the same among seropositiveAGMs and CDC class II HIV-1 patients, although the titer ofinfectious virus in the plasma of SIVagm-infected AGMs waslower than titers in asymptomatic patients. The successfulisolation of SIVagm from the plasma of some animals and thefact that fresh PBMC of these animals were actively produc-ing virus indicate that there is no complete restriction ofvirus replication that could account for the failure of AGMsto develop disease. Having shown that SIVagm replication isapparently not controlled by a vigorous immune response(22), that the degree of in vivo variability is similar to that ofHIV-1 in humans (2), and now that the in vivo viral load iscomparable to that of HIV-1 in asymptomatic patients, thereason for the failure of this lentivirus to induce immuno-suppressive disease in its natural host remains unclear.However, it must be remembered that the periods of highlevels of viremia associated with AIDS have never beenobserved in AGMs, and it is still unknown whether SIVagm,if it ever achieved the titers common for HIV-1 in patientswith AIDS, would also be immunosuppressive.

ACKNOWLEDGMENTS

We thank Sylvia Raupp, Petra Bourquin, and Stephanie Buglerfor their excellent technical assistance.

Part of this work was supported by a grant from the FederalMinistry of Health.

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