Vol. 66, No. 4

JOURNAL OF VIROLOGY, Apr. 1992, p. 2143-2149

0022-538X/92/042143-07$02.00/0 Copyright C 1992, American Society for Microbiology

Quantitation of a Lentivirus in Its Natural Host: Simian Immunodeficiency 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)-infected African 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 blood mononuclear cells (PBMC) of each of nine seropositive animals. The virus DNA load was variable among the monkeys tested, ranging from 5 to 50 (mean = 15) copies per 105 PBMC, which is comparable to that of human immunodeficiency virus type 1 (HIV-1) in humans. The level of infectious SIVagm in PBMC was measured by endpoint 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 infectious doses per 106 cells, similar to the titer shown for HIV in asymptomatic carriers. Free infectious virus was isolated from the plasma of 4 of 17 monkeys (24%), and SIVag expression in peripheral blood in vivo, as demonstrated by in situ hybridization, was detectable only in those animals which were viremic. SIVgm replication is therefore not totally suppressed in vivo, and SIVga has a viral load equivalent to that seen for HIV-1 in asymptomatic humans.

100,000 PBMC produced infectious SIV. m. The detection of viremia in 4 of the 17 plasma sampfes tested and the demonstration of viral mRNA expression in unstimulated PBMC from the viremic animals indicate that AGMs undergo infection with an actively replicating lentivirus.

Simian immunodeficiency viruses (SIV) have been isolated from a variety of different primate species. They represent the closest known relatives of human immunodeficiency virus (HIV) on the basis of their genetic, biological, and antigenic similarities. Therefore, these viruses are suitable as a model for studying the pathogenesis of HIV infection as well as performing extensive vaccination and challenge experiments (18). SIV isolated from African green monkeys (AGMs) (SIVagm) (5, 10, 12, 17, 23) has two properties which make it particularly interesting: first, the AGMs represent the natural host of the virus, as demonstrated by the presence of virus-specific antibodies in a large proportion of wild animals (15), and second, SIVagm induces no disease in its natural host. One possible reason for the failure of SIVagm to induce an AIDS-like disease could be the nature or strength of the immune response in infected AGMs. However, in a recent report we showed that the functional immune response of AGMs to SIVagm is very similar to the response of humans to HIV (22). Moreover, we find that the degree of sequence variation in vivo following infection of AGMs with molecularly cloned virus is comparable to that reported for HIV (2). Another possible explanation of why SIVag-infected AGMs remain healthy could be that infection of T cells in vivo does not result in a productive virus infection. For HIV type 1 (HIV-1), the finding of infectious virus in the plasma of both symptomatic and asymptomatic patients (7, 9, 13) shows that replication in the host is not completely suppressed. In this article, we report that the viral load in the peripheral blood of infected AGMs is comparable to that seen in the blood of HIV-1-infected patients. By using polymerase chain reaction (PCR), SIVagn, sequences were detected in 100% of DNA samples from seropositive AGMs (5 to 50 copies per 105 peripheral blood mononuclear cells [PBMC]), and we found that in infected monkeys, approximately 1 in *

MATERIALS AND METHODS

Animals. Blood was obtained from 21 adult AGMs (8 to 12 old) which had been imported from Ethiopia and Kenya during the years 1978 to 1982 to the primate colony at the Paul Ehrlich Institute. Of these animals, 16 displayed a long-term (>6-year) natural infection by SIVagm, as judged by their prolonged seropositivity; one seroconverted in 1988, and four seronegative AGMs served as controls. All animals were 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 established in our laboratory. PBMC were obtained by FicollHypaque density gradient centrifugation of whole blood. The PBMC layer was removed and washed twice in phosphate-buffered saline (PBS). Cells for use in PCR studies were 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 phytohemagglutinin (PHA) per ml and cocultivated directly with 2 x 106 Molt 4/8 cells in RPMI 1640 with 20% fetal calf serum (FCS) and antibiotics. Coculture supernatants were subsequently monitored weekly for the presence of reverse transcriptase (RT) activity for 8 weeks. During this period, the medium was replaced by fresh medium (RPMI 1640 with 10% FCS and antibiotics) every 2 to 3 days. The lowest number of PBMC which resulted in a positive culture was taken as the endpoint, and the titers of infectious SIVagm were then expressed as tissue culture infectious doses (TCID) per 106 PBMC. In parallel, 107 PBMC were stimulated with 200 ,ug of PHA years

Corresponding author. 2143

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HARTUNG ET AL.

per ml for 24 h and cultured in medium (RPMI 1640 with 20% FCS and antibiotics) containing 10% interleukin-2 (Biotest GmbH, Dreieich, Germany). After 4 days, these cells were cocultivated with Molt 4/8 cells. Plasma culture. After Ficoll-Hypaque centrifugation, the plasma, diluted 1:2 with PBS, was removed and centrifuged at 3,000 x g for 20 min. Subsequently, 10, 1, 0.1, and 0.01 ml of plasma, containing 10 IU of heparin per ml, were each added to 2 x 106 Molt 4/8 cells. Following incubation for 1 h at 37°C, cells were centrifuged to remove the plasma and maintained in culture for up to 8 weeks. During this period, cultures were monitored weekly for the presence of RT activity and medium was replenished every 2 to 3 days. The plasma SIVagm titer was expressed as the number of TCID per milliliter of plasma. To test the sensitivity of the isolation procedure and to provide an internal control, 10 and 1 50% TCID (TCID501 from a precisely titrated virus stock (22) of SIVagm (10 were each added to 10 ml of plasma from a TCID50/ml) seronegative AGM containing 10 IU of heparin per ml (four replicates per dilution). After incubation of the plasma with 2 x 106 Molt 4/8 cells for 1 h at 37°C, cells were centrifuged and 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 (purified disrupted SIVagm) by using standard enzyme-linked immunosorbent assay (ELISA) protocols (19). Briefly, sucrose gradient-purified SIVagm, disrupted in 1% Nonidet P-40 and diluted in water to 20 ,ug/ml, was dried overnight onto flexible microtiter plates (50 ,ul per well). Plates were washed and incubated with blocking buffer (PBS, 3% bovine serum albumin, 10% FCS) before further washing and addition of sera diluted in blocking buffer plus 0.1% Tween 20. After incubation for 1 h at 37°C, the plates were washed and incubated with goat anti-human immunoglobulin G peroxidase conjugate (Sigma) diluted 1:500. Finally plates were washed and incubated with substrate buffer (1 mg of o-phenylenediamine per ml of PBS [pH 6] plus 0.025% H202) before the reaction was stopped with 1 M H2SO4 and optical density at 492 nm (OD492) was measured. Titers were defined as serum dilutions giving an OD that was 50% of the maximum 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 GTG TCT TTC ACT T 3') and (ii) SAGpoI2947+ (5' CCA CCT TAT GAG TGG ATG GGA T 3') and SAGpol4207- (5' CTT CTA TGA ATC CAC TGG CTA C 3'). For detection of the amplified products, a full-length EcoRI insert of cloned SIVagm3 (4) 32p labeled by nick translation was applied during Southern hybridization. For in situ hybridization, a long terminal repeat-derived 35S-labeled RNA probe was prepared by in vitro transcription of an antisense-strand EcoRI-XhoI insert of clone SIVagm3 in the Bluescript plasmid (3) and alkali hydrolyzed as described previously (8). The 2.5-kbp-long fragment spanned the 3' part of the genome, including one complete long terminal repeat. PCR. DNA from 107 PBMC was prepared according to standard methods involving lysis of the cells in the presence of sodium dodecyl sulfate, digestion with proteinase K at 56°C for 12 h, and phenol, phenol-chloroform, and chloroform extractions. After ethanol precipitation in the presence of 0.3 M sodium acetate and centrifugation, the DNA was washed with 70% ethanol, redissolved in water, and quanti-

J. VIROL.

fied by measuring OD260. The DNA was melted at 94°C for 10 min and rapidly cooled to 15°C. The PCR was performed as 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 containing S puM each primer, 200 puM each deoxynucleoside triphosphate, 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl2, 0.01% gelatin, and 2.5 U of Taq polymerase. Subsequently, 30 to 35 cycles of denaturation (94°C; 45 s), hybridization (45°C; 1.5 min), and extension (72°C; 1.5 min) were performed. Complete reactions were separated by size by using 1.2% agarose electrophoresis, and the subsequent Southern analysis was performed by using standard procedures (26). For the standard curve, dilutions of cloned SIVagm3 DNA (in linearized plasmid form) were mixed with 1 pug of carrier DNA from Molt 4/8 cells and analyzed as described above. In situ hybridization. Cells were fixed with 4% formaldehyde in PBS, dried on Silan-coated slides (24), and dehydrated in a graded series of ethanol. Cell pretreatment and in situ hybridization were performed according to the method of Cox et al. (8). The 3 S-labeled RNA probe (prepared as described above) was diluted to about 1 ptg/ml in hybridization buffer containing 50% formamide, 0.6 M NaCl, lx Denhardt's solution, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.5 mg of tRNA per ml, and 100 pug of poly(A) RNA per ml. Hybridization was performed overnight at 55°C. The slides were then rinsed for 24 h at 55°C in 50% formamide-10 mM Tris-HCI (pH 7.5)-i mM EDTA-0.6 M NaCl. Controls were prepared by the same procedure with the sense strand of our probe or by pretreating the slides with 40 ,ug of RNase A per ml for 20 min prior to hybridization. For autoradiographic detection of hybridized RNA, the sections were covered with Kodak nuclear emulsion NTB-2. After exposure for 2 to 14 days, the slides were developed in Dectol (Kodak), fixed, stained with hematoxylin-eosin, and examined with a Zeiss Axiophot microscope. RESULTS Determination of SIV2gm DNA copy number. To quantitate the number of SIVagm DNA copies present in the PBMC of infected AGMs, PBMC lysates from nine seropositive monkeys were subjected to PCR. By using two different oligonucleotide primer pairs specific for the gag and pol genes of all known molecular clones of SIVagm (3, 11, 14), viral sequences were detected in the DNA of all nine animals. Southern analysis of the amplificates with molecular virus clone SIVagm3 as the radioactive probe resulted in specific bands of 0.5 and 1.2 kbp for the gag and pol genes, respectively. In order to estimate the number of viral DNA copies present in PBMC of infected monkeys, the signal intensities from all amplifications were compared by densitometer scanning with the signal intensity resulting from amplification of 101 to 104 copies of a molecularly cloned SIVa m3 DNA. Because of variability of the pol-specific signal (resulting presumably from differences in the degree of homology between the various viruses and the primers used), the gag-specific signal was used throughout for quantification. As shown in Fig. 1, as few as 10 copies of standard viral DNA were detected, and there was a linear relationship between the signal intensity and the amount of viral DNA present in the reaction. In the analysis of the PBMC, the number of SIVagm DNA copies ranged from 5 (by extrapolation of the standard curve) to 50 per 105 cells in different monkeys (Table 1).

SIVagm VIRUS LOAD

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TABLE 1. In vivo virus load of SIVagm

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Animal TCID/106 TCID/ml no. PBMCI of plasmab

5 13 32 pol

_ *db

Z

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gag

b

Signal Intcnsitv*

0 0 0

0 0 0

Viral DNA

(no.i0-1ofcells)C copies!

No. of RNA-

Serum

expressing cells/ titere 106 PBMCd

0

0

-

0