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Journal of Virology, October 2003, p. 10751-10759, Vol. 77, No. 20
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.20.10751-10759.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Persistence and Dissemination of Simian Retrovirus Type 2 DNA in Relation to Viremia, Seroresponse, and Experimental Transmissibility in Macaca fascicularis

Roseanne C. Wilkinson,1* Claire K. Murrell,1 Rebecca Guy,1 Gail Davis,1 Joanna M. Hall,2 David C. North,2 Nicola J. Rose,1 and Neil Almond1

Division of Retrovirology,1 Division of Virology, National Institute for Biological Standards and Control, Potters Bar, Hertfordshire EN6 3QG, United Kingdom2

Received 8 April 2003/ Accepted 14 July 2003


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ABSTRACT
 
Endemic simian retrovirus (SRV) infection can cause fatal simian AIDS in Macaca fascicularis, but many individuals survive with few clinical signs. To further clarify the parameters of SRV pathogenesis, we investigated the persistence of viral DNA forms in relation to active viremia, antibody response, and transmissibility of infection. In M. fascicularis from endemically SRV-2-infected colonies, viral DNA was present in both linear and unintegrated long terminal repeat circular forms in peripheral blood mononuclear cells of all viremic and many nonviremic animals. Long-term followup of three individuals with distinct infection patterns demonstrated persistence of linear and circular forms of viral DNA in peripheral blood mononuclear cells and tissues, irrespective of viremia or antibody status, but reactivation of latent infections was not observed. The role of viral DNA in transmission and early pathogenesis of SRV-2 was investigated by inoculation of SRV-2 DNA-positive blood into groups of naïve M. fascicularis from either a viremic or nonviremic donor and subsequent analysis of the virological and serological status of the recipients. Transmission of SRV and development of anti-SRV antibodies were only observed in recipients of blood from the viremic donor; transfer of SRV provirus and unintegrated circular DNA in blood from the nonviremic donor did not lead to infection of the recipients. These results indicate that a proportion of M. fascicularis are able to effectively control the replication and infectivity of SRV despite long-term persistence of viral DNA forms in infected lymphocytes.


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INTRODUCTION
 
Simian retrovirus (SRV) is a Betaretrovirus capable of causing an AIDS-like disease in simians (6, 10-12). Rapidly spread through bodily fluids (17, 22, 34), SRV causes an endemic infection of wild (23a) and captive (12, 17) simian colonies and offers a model of host-retrovirus interactions. Unlike experimental infection of macaques with wild-type simian immunodeficiency virus (SIV), infection with SRV can produce a range of clinical outcomes (11, 12, 15, 22). Anti-SRV antibody responses are mounted in a proportion of individuals and correlate with a decline in viremia (15). Some individuals appear to eliminate the infecting virus, while others survive with chronic low-level viremia (11, 12, 15, 22). Individuals that do not produce effective antibodies develop disease, which can be fatal. To clarify the underlying mechanisms of host-virus interaction that lead to these different outcomes, we sought to investigate further the parameters of SRV-2 pathogenesis.

Molecular diagnostic techniques have proved valuable to characterize the pathogenesis of human immunodeficiency virus (HIV) and SIV infections (1, 4, 23, 30). To complement virological and serological techniques in characterization of SRV infection, we have developed and applied DNA PCR assays that detect conserved long terminal repeat (LTR) and primer-binding site sequences and unintegrated LTR circles of this virus. Previous studies have shown that, at least in the case of HIV, persistence of provirus can contribute to ongoing replication and infectivity (24). Latent reservoirs of provirus may exist in quiescent T cells that are reactivated on stimulation of those cells to divide. LTR circles are unintegrated DNA species formed during viral replication that may be stable over prolonged periods (3, 25). Accumulation of LTR circular DNA in peripheral blood mononuclear lymphocytes (PBMCs) correlates with progression to AIDS in HIV infection (23).

In this study, we analyzed the relationship between the presence of viral DNA forms and the persistence of infectious viremia and antibody responses in naturally occurring and experimental infections with SRV-2. The DNA PCR assays as well as virus isolation assays were applied in a cross-sectional analysis of SRV-2 infection in a cohort of 67 animals and a longitudinal study of three animals naturally infected with SRV-2. Anti-SRV antibodies in these three individuals were measured by enzyme-linked immunosorbent assay (ELISA). These data led us to investigate the virus-host interactions that may affect transmission of SRV-2 and control of viremia in the early stages of infection. As a result, we carried out a study of experimental transmission in which viral DNA-positive blood from viremic or nonviremic donors was inoculated into naïve recipients. Postinoculation, the infection status of the recipients was followed by DNA PCR, virus isolation, and ELISA.

Information from these studies increases our understanding of the pathogenesis following infection with SRV and allows comparison with what has been observed following infection with SIV or HIV. Comparison of the mechanisms of immune response and viral replication in these systems may help to identify the crucial factors that allow the host control of SRV that is not observed following infection with SIV or HIV. The data may also prove useful in the management of SRV-2 in colonies of nonhuman primates.


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MATERIALS AND METHODS
 
Animals. Purpose-bred cynomolgus macaques (Macaca fascicularis), housed and maintained in accordance with United Kingdom Home Office guidelines for the care and maintenance of nonhuman primates, were used for this study. Before inoculation with virus or venipuncture, macaques were sedated with ketamine hydrochloride.

Study 1. Sixty-seven macaques of undefined SRV status but derived from colonies known to be endemically infected with SRV-2 were screened by virus isolation, ELISA, and diagnostic DNA PCR. A single 6-ml sample of whole blood was collected from all animals into EDTA as an anticoagulant.

Study 2. From the sixty-seven animals screened in study 1, three macaques, 977G, 540FC, and 963DBDA, were followed over a 245-day period; whole blood samples were collected at approximately monthly intervals and analyzed for the presence of SRV by virus isolation and DNA PCR and for anti-SRV antibodies by ELISA.

Study 3. For a study of SRV-2 transmission, four recipient animals, X382, X383, X384, and X385, were selected from colonies known to be free of SRV infection. Recipient animals were inoculated intravenously with 1 ml of whole blood, collected into citrate to a final volume of 10%, from the donor animal. At termination of the transmission study, the animals were killed humanely by overdose of anesthetic, and tissue samples (peripheral lymph node, mesenteric lymph node, lung, thymus, liver, salivary gland, salivary gland lymph node, spleen, bone marrow, large intestine, small intestine, brain, and cerebellum) were collected from the donor and recipient animals.

Cell culture and SRV propagation. Raji cells (8) were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 1% penicillin, and 1% streptomycin. Serogroup-defined SRV-1, SRV-2, SRV-3, SRV-4, and SRV-5 virus isolates, as referenced (31), were propagated in Raji cell cultures.

Virus isolation and plasma sample preparation. Whole blood (3 ml) was diluted 1:3 with RPMI 1640 medium and fractionated by density gradient centrifugation with 55% Percoll (Sigma, Poole, United Kingdom) according to the manufacturer's conditions. Plasma was heat inactivated. Peripheral blood mononuclear cells (PBMCs) were collected, washed twice in RPMI 1640 medium, and enumerated. These PBMCs were further diluted in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 1% penicillin, and 1% streptomycin. Either 5 x 106, 5 x 105 or 5 x 104 PBMCs were cocultured with 105 freshly passaged Raji cells. Cultures were fed and inspected every 3 or 4 days over 14 days for syncytium formation in SRV-positive cultures in comparison with uninfected Raji cells.

DNA extraction. Blood (1 ml) or homogenized tissue was added to 9 ml of red blood cell lysis buffer (154 mM NH4Cl, 10 mM KCl, 0.8 mM tetrasodium EDTA), and white blood cells were pelleted by centrifugation (800 x g for 5 min). The cell pellet was resuspended in 10 mM Tris, pH 8.0, 50 mM NaCl, and 10 mM EDTA (TNE) containing 0.25% sodium dodecyl sulfate and proteinase K (Sigma; final concentration, 25 µg/ml) and incubated overnight at 56°C. Following phenol-chloroform extraction and alcohol precipitation as previously described (27), DNA was resuspended in 200 µl of nuclease-free water. Extraction controls (no biological material) were prepared in parallel.

PCR amplification. PCR primers DLTRN, DCTEN, DPBSC, D2LTRN, and D2LTRC and hybridization probes DLTRprobe and D2LTRprobe were designed by selection of conserved sequences from the LTR, primer-binding site, and constitutive transport element regions of the SRV genome following alignment of the SRV-1, SRV-2, and SRV-3 genomes with GCG sequence analysis software package (7). These oligonucleotides had little homology with sequence from a known endogenous SRV-like genome (35). A 277-bp LTR fragment was amplified with sense oligonucleotide DLTRN (5'-TTTCCCGCCGGCGCGAATATTTCC3', SRV-2 nucleotide positions 103 to 126, accession number M16605) (33) and antisense oligonucleotide DPBSC (5'-TTCCCTCGTATCCAGCCCCACGTT-3', SRV-2 nucleotide positions 380 to 357). DLTRN recognizes a sequence in the LTR, and DPBSC recognizes the primer binding site of SRV-1 (accession number M11841) (26), SRV-2 and SRV-3 (accession number M12349) (32). A 449-bp one-LTR circle fragment was amplified with DPBSC and sense oligonucleotide DCTEN (5'-TGTATCACTCCAACCTAAGACAGGC-3', SRV-2 positions 7690 to 7714), which recognizes sequence in the constitutive transport element of SRV-1, SRV-2, and SRV-3.

A 215-bp two-LTR circle fragment was amplified across the LTR junction with sense oligonucleotide D2LTRN (5'-TGT CTT GTC TCC ATT TCT TGT-3', SRV-2 positions 257 to 277 in the U5 region) and antisense oligonucleotide D2LTRC (5'-GGAAATATTCGCGCCGGCGGGAAA-3', SRV-2 positions 126 to 103 in the U3 region). D2LTRN and D2LTRC recognize sequences in SRV-1, SRV-2, and SRV-3.

A 440-bp envelope fragment was amplified with previously reported oligonucleotides specific for SRV-2 (9). These were sense oligonucleotide SRV2E6247N (5'-ATGTCCCCACAGTTTTGG-3') and antisense oligonucleotide SRV2E6687C (5'-TAAATTCAAGAGGTTGTACCAACAAAG-3'). Envelope fragments were amplified specifically from SRV-1 and SRV-3 DNA, with previously described PCR methods (36). DNA amplification was performed in a T3 thermal cycler (Hybaid, Teddington, United Kingdom). DNA (10 µl) was amplified in a 50-µl reaction (final volume) comprising 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 2, 1.5, 2, or 2.5 mM MgCl2 for LTR, one-LTR circle, two-LTR circle, and env assays, respectively; 0.25 mM each oligonucleotide; 0.2 mM each dATP, dCTP, dGTP. and dTTP; and 1.25 U of Amplitaq Gold DNA polymerase (Applied Biosystems, Warrington, United Kingdom).

Amplification for the LTR and two-LTR circle assays consisted of a single cycle at 94°C for 10 min, followed by 40 cycles comprising 15 s at 94°C, 15 s at 55°C, and 30 s at 72°C. Amplification for the one-LTR circle and env assays consisted of a single cycle at 94°C for 10 min, followed by 40 cycles comprising 1 min at 94°C, 2 min at 55°C, and 3 min at 72°C. Each amplification reaction was ended with a 10-min incubation at 72°C. Amplified products were analyzed by electrophoresis through 1.25% agarose gels and visualized by UV illumination after treatment with ethidium bromide. Southern hybridization with sequence-specific probes was used to confirm that amplification products were derived from SRV sequences. For both LTR and one-LTR circle assays, hybridization was performed with DLTRprobe (5'-ACAAGAAATGGAGACAAGACA-3', SRV-2 positions 277 to 257), which recognizes SRV-1, SRV-2, and SRV-3. For the two-LTR circle assay, hybridization was performed with D2LTRprobe (5'-TGTCCCGACCCGCGGGA-3', SRV-2 positions 346 to 330), which recognizes SRV-1, SRV-2, and SRV-3. For the env assay, SRV2E6547N (5'-CTAATTTGGCCAACGANT-3') was used, which is SRV-2 specific.

Analysis of PCR endpoint dilution assays. In view of differential detection of SRV DNA by the PCR assays in certain samples, the relative sensitivity of each assay was investigated by determining the endpoint of detection in serial dilutions of a known SRV-positive sample. Analysis of the endpoints of detection was also performed to compare proviral loads in donated blood of macaques 977G and 540FC and for determination of the limit of detection of SRV in animals X382 and X383. Each dilution was replicated five times. The number of viral DNA copies in the undiluted sample was estimated where required, according to the single-hit Poisson statistical model (5). In this model, the number r of positive samples out of n tested at each dilution follows a binomial distribution, with mean E(r/n) = P = 1 - exp (-Xz), where p is the probability of a positive sample, X represents the total copies per volume of the undiluted sample, and z is equal to sample volume/dilution factor.

Lysate preparation and ELISA detection of specific antibodies. To prepare virus lysate to use as an ELISA antigen, 100 ml of Raji cells (2 x 106 cells/ml) was infected with supernatant from SRV-infected cocultures of PBMCs derived from macaque 977G. When syncytium formation was observed, the Raji cells were pelleted and resuspended in 2 ml of 0.5% (vol/vol) Nonidet P40. Cell lysates were similarly prepared from uninfected Raji cells as controls. The wells of 96-well plates were coated with either 1 µl of virus lysate or uninfected Raji cell lysate in 50 µl of distilled water and dried overnight at 37°C. Nonspecific protein-binding sites were blocked for 30 min with phosphate-buffered saline containing 5% (vol/vol) porcine serum and 0.05% (vol/vol) Tween 20. Following five washes with phosphate-buffered saline containing 0.05% (vol/vol) Tween 20, plasma samples were titrated in threefold dilutions from an initial 1:30 dilution and allowed to bind for 1 h at room temperature. Negative control samples from animals derived from an SRV-free colony were included on each plate.

After washing with phosphate-buffered saline, specific bound antibodies were detected with affinity-purified goat anti-human immunoglobulin coupled to horseradish peroxidase (Sigma) with 3,3,5,5-tetramethylbenzidine as the substrate. The A450 was measured with a Labsystems Multiskan MS plate reader (Thermo Life Sciences, Basingstoke, United Kingdom). A cutoff absorbance value was determined as the mean absorbance value for two negative control plasma samples plus 3 standard deviations (equivalent to 1.5 log10 endpoint titer due to a 1:30 starting dilution of the plasma samples). Results were expressed as log10 endpoint titers determined by linear regression analysis. A sample was considered positive when the log10 endpoint titer with the infected cell lysate antigen was greater than 1.5. Seroreactivity to uninfected Raji cell lysates was analyzed in parallel to detect antibody binding to cellular components in the lysates and was negligible. Comparison of pre- and post-SRV infection plasma samples from macaques X382 and X383 confirmed that the ELISA with SRV-infected cell lysate antigen was capable of specifically detecting antibodies produced as a result of exposure to SRV.

Western blot analysis of plasma antibody reactivity to viral proteins. The proteins within lysates prepared from SRV (977G isolate)-infected Raji cells or uninfected Raji cells were denatured in Laemmli's sample buffer and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) through a 12% gel with Mini-Protean II gel apparatus (Bio-Rad, Hemel Hempstead, Hertfordshire, United Kingdom). Proteins were immobilized on nitrocellulose membrane by electrotransfer with the Mini-Protean II transfer tank (Bio-Rad). Immobilized proteins were incubated for 1 h with plasma samples diluted 1:200 in phosphate-buffered saline containing 0.1% (vol/vol) Tween 20 and 5% Marvel nonfat milk powder with the Mini-Protean II multiscreen apparatus (Bio-Rad). to analyze multiple plasma samples simultaneously. The membrane was washed three times for 15 min each in phosphate-buffered saline containing 0.1% Tween 20 before 1 h of incubation with goat anti-human immunoglobulin G antiserum conjugated with horseradish peroxidase (Sigma, United Kingdom), diluted 1:10,000 in phosphate-buffered saline containing 0.1% (vol/vol) Tween 20 and 5% Marvel nonfat milk powder. The membrane was washed as described previously, and antibody reactivity was visualized by enhanced chemiluminescence (ECL; Amersham, Chalfont St. Giles, Bucks, United Kingdom).


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RESULTS
 
Design and evaluation of PCR assays. To reduce the number of false-negative or false-positive results observed by PCR detection of SRV infections, a novel PCR assay was designed to detect areas of the LTR region and the primer-binding site that are well conserved among exogenous SRV genomes. To evaluate the specificity of primers DLTRN and DPBSC, a PCR assay (the LTR assay) was performed with DNA extracted from Raji cells infected with SRV-1, SRV-2, SRV-3, SRV-4, or SRV-5. Specificity of PCR amplification was confirmed by Southern analysis with a probe that detects SRV-1, SRV-2, and SRV-3 (DLTRprobe; Fig. 1A). The LTR assay was also performed on DNA extracted from the PBMCs of SRV-uninfected macaque colonies (Fig. 1A). The LTR assay primers specifically amplified sequences of SRV-1, SRV-2, and SRV-3, as expected, but not SRV-4 or SRV-5; no fragments of the correct size were amplified in control DNA from uninfected macaques.



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  		FIG. 1. Analysis of SRV-specific PCR products by Southern hybridization. Samples were analyzed by both (A) LTR assay (277-bp product) and (B) one-LTR circle assay (449-bp product). Samples were: H2O (lanes 1 and 2), extraction control (lane 3), DNA extracted from Raji cells infected with SRV-1 (lane 4) or SRV-2 (lane 5), uninfected Raji cell control (lane 6), extraction control (lane 7), DNA extracted from Raji cells infected with SRV-4 (lane 8) or SRV-5 (lane 9), extraction control (lane 10), DNA extracted from Raji cells infected with SRV-3 (lane 11), DNA from uninfected macaque PBMCs (lanes 12 to 23), extraction control (lane 24), DNA from infected macaque PBMCs (lane 25), H2O (lane 26).

A second PCR assay was designed to detect unintegrated one-LTR circles of SRV proviral DNA in SRV-infected macaque PBMCs in order to determine whether the presence of this species of viral DNA correlated with the activity of an infection. Oligonucleotides were designed to amplify between conserved regions of the 3' constitutive transport element and the primer-binding site (DCTEN and DPBSC, respectively) of SRV-1, SRV-2, and SRV-3. This assay was designated the one-LTR circle assay. The ability of these oligonucleotides to amplify SRV-1, SRV-2, SRV-3, SRV-4, or SRV-5 is shown in Fig. 1B. The specificity of the PCR amplification was confirmed by Southern analysis with DLTRprobe. SRV-1, SRV-2, and SRV-3, as expected, and SRV-5 PCRs yielded a positive signal with these primers, whereas SRV-4 was not amplified. SRV-specific one-LTR circles were not detected in DNA extracted from PBMCs of macaques derived from SRV-free colonies (Fig. 1B). PCR designed to amplify two-LTR circles from SRV-infected macaques yielded very low quantities of product in a minority of samples, although fragments of the correct size could be readily amplified from DNA extracted from SRV-infected Raji cells (data not shown).

DNA amplification with serogroup-specific oligonucleotides had previously determined macaques within our colonies to be infected with SRV-2 (28). As a result, the previously described SRV-2-specific env PCR assay (9) was used in parallel with the LTR and one-LTR circle DNA PCR assays for analysis of DNA samples. During the evaluation of these primers in cross-sectional analyses, some macaques were observed to be virus isolation positive and env negative; to confirm that these were not infected with other common SRV serogroups, DNA from one such individual, 977G, was also tested with alternative PCR assays that are reported to specifically amplify SRV-1 and SRV-3 env sequences (36). These assays failed to amplify any specific signals from this DNA, although each assay could detect, specifically, positive control DNA isolated from Raji cells infected with the respective virus serogroups (data not shown).

The endpoints of detection of SRV DNA by the LTR, one-LTR circle and env PCR assays were compared by analyses of serial dilutions. Although, in theory, the one-LTR circle assay detects only circular viral DNA and the LTR and env assays are capable of detecting circular and linear species, the one-LTR circle assay was observed to be approximately 2-fold and 10-fold more sensitive than the env and LTR assays, respectively (data not shown).

Cross-sectional analysis of the virological status of cynomolgus macaques derived from colonies endemically infected with SRV. To characterize the range of patterns of infection, sixty-seven macaques were investigated for SRV infection by virus isolation by coculture with Raji cells. Of these, sixteen (24%) were positive (Table 1). To complement diagnostic virus isolation, the LTR and the one-LTR circle DNA PCR assays were applied to DNA isolated from each of the sixty-seven macaques. These data were compared with the virus isolation data and are presented in Table 1. The SRV-2-specific env PCR assay (9) was also performed on each DNA sample (Table 1). Individually, each PCR assay determined a greater number of macaques to be infected than were detected by virus isolation. Seven macaques were determined to be positive by virus isolation, LTR and one-LTR circle assays but negative by the env assay. Nine were determined positive by the LTR and env assays but negative by the one-LTR circle assay and virus isolation assay. The presence of one-LTR circles was not limited to samples that were virus isolation positive; 20 subjects were negative for virus isolation but positive by all three PCR assays. Five macaques were positive by the one-LTR circle assay only.


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  		TABLE 1. Virus isolation and PCR analysis of SRV infection of cynomolgus macaque PBMC samples

Categorization of SRV infection status of three macaques: viremia and antibody response. Three of the macaques from the cross sectional studies were selected for a longitudinal analysis of SRV status. PBMC samples were analyzed over a 245-day period from three SRV-infected macaques by virus isolation, DNA PCR, and serology. Virus isolation and PCR assay results were compared for each sample (Table 2). 977G was consistently virus isolation positive, LTR and one-LTR circle assay positive, and negative by the env assay. 540FC was virus isolation negative but positive by all three PCR assays at every time point. 963DBDA was consistently virus isolation negative but intermittently PCR positive for SRV sequences. One sample from 963DBDA was env assay positive but negative by other PCR assays.


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  		TABLE 2. Longitudinal analysis of PBMC infection status by virus isolation and PCRa

To confirm that PBMCs of 540FC and 963DBDA were not infected with nonsyncytium-forming SRV, LTR and one-LTR circle PCR assays were performed on DNA extracted from the virus isolation cultures at the final time point. Only 977G cocultures were DNA PCR positive (data not shown). To further characterize the infection status of these three macaques, anti-SRV antibody responses were determined by ELISA with whole cell lysate antigen prepared from Raji cells infected with virus isolated from 977G. The results are presented in Fig. 2A. Plasma from 977G had low levels of anti-SRV antibodies; log10 endpoint titers never exceeded 2.3. By contrast, 540FC and 963DBDA had strong persistent serological responses, measuring between 3.0 and 3.6 log10 endpoint titer. Intriguingly, 963DBDA had lower seroreactivity to SRV at the initial time point (log10 endpoint titer 2.1).



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  		FIG. 2. Analysis of serum anti-SRV antibodies by ELISA. 977G viral lysate was used as the antigen substrate. ELISAs were performed in duplicate, and the graphs shown are representative. A log10 endpoint titer of >1.5 was considered a positive result; the cutoff is indicated by the dashed line. (A) Sequential analysis of anti-SRV antibodies in three animals, ({blacksquare}) 540FC, ({triangleup}) 963DBDA, and ({diamondsuit}) 977G. (B) Analysis of recipient anti-SRV antibodies. Group A recipients of blood from 977G: ({diamondsuit}) X382, {square} X383. Group B recipients of blood from 540FC: ({triangleup}) X384, ({blacksquare}) X385.

Study to evaluate transmission of SRV. To establish the relative ease of transmission of SRV from virus isolation positive and DNA PCR positive or virus isolation negative and DNA PCR positive macaques, 1 ml of citrated blood from 977G or 540FC was transferred by intravenous inoculation to pairs of macaques derived from colonies known to be SRV-free. Furthermore, each recipient individual had been demonstrated to be virus coculture negative and DNA PCR negative by LTR and one-LTR circle PCR on two occasions prior to challenge. Recipients X382 and X383 (group A), received blood from 977G; recipients X384 and X385 (group B) received blood from 540FC. Enumeration of PBMCs collected for virus isolation from each donor animal on the day of inoculation indicated that 1 ml of whole blood contained at least 5 x 106 PBMCs. Endpoint dilution LTR assays performed on DNA extracted from 977G and 540FC blood on the day of challenge indicated that 977G had a sevenfold greater SRV proviral load than 540FC (approximately 2,000 and 300 viral copies/106 cells, respectively; data not shown).

At selected times up to 229 days following challenge, virus isolation, DNA PCR, and ELISA were performed on blood collected from each recipient. No evidence of SRV infection was detected by virus isolation, PCR, or ELISA in blood from X384 and X385 that were inoculated with blood from 540FC (Table 3; Fig. 2B). In contrast, both X382 and X383 that received blood from 977G were virus isolation positive and one-LTR circle positive at day 14 (Table 3). At day 28, X382 and X383 were virus isolation negative and remained virus isolation negative until the end of the study; however, one-LTR circles persisted, intermittently, in both animals.


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  		TABLE 3. Virus isolation and PCR analysis of SRV infection in recipient cynomolgus macaque PBMCsa

To determine whether the differential ability to detect SRV DNA by the LTR and one-LTR circle assays reflected a low proviral burden in the blood, one-LTR circle PCR was performed on serial dilutions of DNA from X382 and X383 collected at day 229 (data not shown). The dilution endpoints of detection for the X382 and X383 samples were 10-1 and 10-2, respectively. X382 and X383 were seronegative prior to inoculation, but by day 28, strong anti-SRV antibody responses were detected by ELISA (Fig. 2B). These responses persisted thereafter at approximately 3.0 log10 endpoint titer, until the end of the study.

Anti-SRV antibody reactivity in plasma samples of X382 and X383 at days -21 and 109 was characterized further by Western blot analysis with whole-cell lysate antigen prepared from SRV-infected or uninfected Raji cells (Fig. 3). Parallel Western blot analysis of anti-SRV antibody reactivity in plasma samples from blood donors 977G and 540FC was also performed for comparison. No immunogenic response was detectable in plasma samples prepared from X382 or X383 at day -21 before inoculation with SRV-infected blood. At day 109 postinoculation, plasma antibodies from X382 and X383 showed reactivity to protein at 70 kDa. Plasma antibodies from X382 also showed weak reactivity to proteins between 20 kDa and 35 kDa. The viremic 977G showed no detectable antibody reactivity whereas the nonviremic, DNA PCR-positive 540FC displayed the strongest antibody reactivity, towards viral proteins at 70 kDa and between 20 kDa and 35 kDa. The 70-kDa protein corresponds in molecular size to SRV envelope protein gp70. SRV proteins with molecular masses of between 20 kDa and 35 kDa include the envelope protein gp20 and the Gag protein p27. A low degree of reactivity was detected, at approximately 60 kDa, to the uninfected as well as SRV-infected cell lysates for plasma samples from infected animals.



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  		FIG. 3. Western blot analysis of plasma anti-SRV antibody reactivity. Proteins within the lysates prepared from Raji cells infected with the 977G SRV isolate or uninfected Raji cells were separated by SDS-PAGE and analyzed by Western blotting with the following plasma samples: X382 day -21 prebleed (lane 1), X382 day 109 postinoculation (lane 2), X383 day -21 prebleed (lane 3), X383 day 109 postinoculation (lane 4), 977G day 168 (lane 5), and 540FC day 168 (lane 6). Viral proteins were detected by plasma antibody reactivity at 70 kDa and between 20 kDa and 35 kDa. A low level of reactivity towards uninfected Raji cell components was detected at approximately 60 kDa.

Distribution of virus in tissues of SRV-infected macaques. To establish whether diagnostic DNA PCR on DNA extracted from whole blood provided an accurate and sensitive detection of SRV infection, LTR and one-LTR circle assays were performed on DNA extracted from selected tissues of 977G, 540FC, 963DBDA, X382, X383, X384, and X385. The results of these studies are presented in Table 4. 977G displayed the widest distribution of SRV, with every tissue examined testing positive by the LTR and one-LTR circle assays, except for brain samples, which were positive by the one-LTR circle assay only. 540FC had a wide distribution of SRV-infected tissues; DNA PCR failed to detect a specific signal in the lung only, although several tissues were positive by one assay only. 963DBDA displayed a more restricted distribution of SRV within lymphoid tissues. SRV was detected only by the one-LTR circle assay in lymphoid tissues from X382 and X383. No evidence of SRV infection was found in the tissues examined from X384 and X385.


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  		TABLE 4. Tissue distribution of SRV proviral DNA as measured by the LTR and 1 LTR circle PCR assaysa

To determine whether the reisolation of virus from the tissues correlated with the ability to recover virus from the PBMCs, lymphocytes from peripheral lymph nodes or mesenteric lymph nodes were cocultivated with Raji indicator cells. Initially, peripheral lymph node samples were taken from 977G, 540FC and 963DBDA to determine whether the coculture of lymph node cells would reveal otherwise occult infection. Since coculture of peripheral lymph node cells did not prove to be more sensitive for the reisolation of SRV, mesenteric lymph nodes of the recipients, X382, X383, X384, and X385, were sampled on termination. Virus was reisolated from the peripheral lymph nodes of 977G only.


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DISCUSSION
 
In this study we have established novel molecular diagnostic assays to complement existing virological and serological methods in an analysis of the relationship between the persistence and dissemination of SRV viral DNA forms and viremia, antibody response and transmissibility of infection. In a cross-sectional analysis of sixty-seven macaque blood samples with DNA PCR assays in parallel with virus isolation assays to investigate virological status, LTR sequences and one-LTR circles were detected in all sixteen viremic animals; 34 nonviremic animals also had linear or circular SRV viral DNA forms in their PBMCs, detected by LTR, env or one-LTR circle PCR assays. This is in accordance with previous studies in which proviral SRV DNA has been detected by PCR in PBMCs of nonviremic animals (16, 29); however, an analysis of the frequency of detection of unintegrated SRV DNA in a cohort of animals has not been performed before.

Long-term follow-up of three individuals showed that each had a distinct infection pattern. Animal 977G was viremic and positive for LTR and one-LTR circle DNA PCR assays while animal 540FC was not detectably viremic and was positive for LTR, env and one-LTR circle DNA PCR assays. Animal 963DBDA also was not detectably viremic and was DNA PCR positive but the observed frequency of positive PCR signals in PBMCs diminished during the course of the sequential bleeds. All three animals had detectable viral DNA within tissues as well as PBMCs, although the extent of dissemination was more limited in 963DBDA; virus was only reisolatable from 977G. The possibility remains that 540FC and 963DBDA may have had very low levels of viremia but the consistently negative virus isolation results for these animals on at least seven occasions strongly indicate that virus activity was well controlled and persistently at a very low load.

These studies show that SRV viral DNA, including unintegrated one-LTR circles, persists in infected individuals even in the long-term absence of detectable viremia. However, the abundance of SRV sequences may decrease over time, possibly due to the death of infected cells, which, in the absence of replicating virus within the tissues, are not replaced by newly infected cells. ELISA data for these three animals indicated that serological responses and viremia are inversely related, as previously noted (15, 28). 977G had a minimal antibody response against SRV, whereas 540FC and 963DBDA had strong antibody responses. Viral DNA species were present in both seropositive and viremic animals.

A number of questions arose from these data. Could inoculation of SRV provirus into unexposed animals lead to reactivation of a latent infection? What are the early events that occur during infection and how do they correlate with disease outcome and dissemination or activity of provirus? We designed a study of SRV transmission in order to address these issues, to increase understanding of the pathogenesis of SRV infections as well as to provide information that would aid effective detection and management of the virus within captive colonies. Blood was inoculated into pairs of naïve macaques, from viral DNA positive donor animals that were viremic or not detectably viremic. Only blood from the viremic donor, 977G, was observed to be infectious, and both recipients of this blood, X382 and X383, became virus isolation and DNA PCR positive within 14 days postinoculation. There was a less than tenfold difference between the proviral load of 540FC and that of 977G; the recipients of 540FC blood, X384 and X385, were inoculated with more than 1,500 copies of SRV DNA sequence in 1 ml of blood (containing at least 5 x 106 PBMCs), yet they did not become infected.

Although we cannot rule out the possibility that X384 and X385 were resistant to SRV-2 infection, the results suggest that provirus within the blood of 540FC was nontransmissible. The early viremia detected in recipients of blood from 977G was short-lived and disappeared by day 28, although viral DNA persisted intermittently in both animals until termination. This viral DNA was present in a variety of lymphoid tissues as well as the PBMCs. Only one-LTR circles were detected, which were low in frequency when tested by dilution assay; this perhaps explains the absence of signals detected by the less sensitive LTR PCR assay. ELISA data also indicated that recipients of 977G blood had been exposed to SRV antigens that induced anti-SRV antibody production by day 28, at the time when viremia ceased, whereas recipients of 540FC blood remained unexposed to antigen and did not produce antibodies. Western blot analyses of anti-SRV immunogenic reactivity in plasma samples of donors and recipients again showed that levels were highest in 540FC and antibodies were also present in X382 and X383 after inoculation but not in plasma from 977G and the naïve recipients. The SRV envelope protein gp70 appears to be the most immunogenic antigen in the animals studied. Antibodies against the other viral proteins of between 20 kDa and 35 kDa were also detected most strongly in 540FC plasma and were weakly detectable in plasma from X382 postinfection.

In our study, we used DNA PCR assays to analyze pathogenesis and have also demonstrated efficient assays that can be used to screen for SRV infection among colonies. We found that the novel LTR PCR assay detected more infections than the previously reported env assay, despite the observation that the env assay was more sensitive in serial dilution assays. Most importantly for diagnostic purposes, the LTR assay detected all viremic animals. It is interesting that almost half of the viremic animals in the cross-sectional study were env assay negative (env PCR assays designed to detect alternative serogroups, SRV-1 and SRV-3, also failed to amplify any sequence in DNA from one such individual); we propose that minor variation in the SRV env sequence of virus of serogroup 2, as has been reported previously (18, 28), could account for this. Viremia and virus replication would be expected to result in the generation of more sequence variants. Alternatively, SRV variants with altered env sequences may have greater potential for replication and evasion of host immunity. The majority of infected animals were positive by both LTR and one-LTR circle assays. Recent studies have indicated that LTR circles of retroviruses can persist for prolonged periods in surviving cells in the absence of virus replication (3, 25), therefore it is not surprising that not all one-LTR circle-positive individuals were viremic. Some individuals were positive by the LTR PCR assay and negative for one-LTR circles. The absence of one-LTR circles in some samples may be due to a loss of circular DNA species through death or division of the cells that harbored them, while integrated provirus would persist in an inactive form, as it would be duplicated each time an infected cell divides. The one-LTR circle assay is more sensitive than the LTR assay, which may explain why some samples were positive for one-LTR circles but LTR sequences were not detected. When used in conjunction, however, these two assays would appear capable of identifying all infected individuals, except those with low proviral loads, which may have inactive infections but could be detectable by serology.

Previous studies of SRV have not clarified the relationship between the presence of viral DNA and the activity or infectivity of the virus. The data in this study suggests that the occurrence of viral DNA, including unintegrated forms, throughout tissues in SRV-infected animals is not inevitably associated with active and infectious viremia. Individuals may effectively control SRV despite the persistence of copies of the viral genome. Abrogation of viremia by the host immune system can occur rapidly after experimental infection as was observed in this and other studies (15); primary viremia may be reduced to undetectable levels within a month in some individuals. Previous studies of SRV also indicate that effective control of virus activity may be long-term in some individuals (11, 15, 20), and reactivation of latent SRV infection was not evident in our study. The nature of the protective immunity involved is unknown and may include cellular or humoral mechanisms, or both.

Production of neutralizing antibodies, which effectively prevent virus particles from infecting their target cells, is likely to play an important role in the response to SRV infection. In support of this theory, successful vaccinations against SRV infection have been reported, which elicited neutralizing antibodies (2, 13, 19). The roles of other adaptive immune processes, such as the action of cytotoxic T lymphocytes, have not been examined. Cellular defense mechanisms may also contribute to maintenance of the inactive state of a retrovirus infection. During latent HIV infection, for example, viral replication is blocked at reverse transcription (14). Control of SRV replication in this manner in the target T and B cells (21) may aid inactivation of the infecting virus, but this has yet to be demonstrated.

Investigation of the different forms of viral DNA that accumulate during retroviral infections may help to identify modes of cellular control of replication that play a role in pathogenesis. PCR assays used in our study detect one-LTR circles but only small quantities of two-LTR circles in vivo. It is intriguing that similar studies of SIV infection have detected two-LTR circles rather than one-LTR circles (4). The molecular processes involved in SRV replication may differ from those involved in SIV infection, favoring recombination events or a block in reverse transcription that would lead to formation of one-LTR circles. Interestingly, in pathogenic SIV infection, two-LTR circles persist in tissues but not PBMCs following primary viremia (4), yet in SRV infection, one-LTR circles are detected in PBMCs and also are widespread among tissues. This detection of alternative species of unintegrated viral DNA in differing locations may reflect host-virus interactions that lead to the differential pathogeneses of SRV and SIV within the same host.

Our investigation of the relationship between SRV-2 viral DNA species, viremia and seroresponse in M. fascicularis shows that SRV-2 DNA can persist even in antibody positive individuals that have no detectable viremia. Effective immune control in such animals may prevent reactivation of the virus and transmission to other animals. A greater proportion of M. fascicularis achieve long-term control of viremia when infected with SRV than when infected with SIV despite this persistence of provirus copies in both types of infection. The seemingly low pathogenicity of SRV-2 in this species may be in part due to the fact that SRV is endemic to the Asian macaques and so a degree of host-virus coadaptation may have occurred over many generations. In contrast, macaques are not the natural host for SIV and succumb to fatal simian AIDS as a result of infection with this virus. SRV infection, resulting in immunodeficiency disease in only a proportion of animals, may therefore be valuable as a model of host-virus interactions to compare with other, both pathogenic and nonpathogenic retrovirus infections, in order to identify the important correlates of pathogenesis.


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ACKNOWLEDGMENTS
 
We are grateful to Paul Clapham (University of Massachusetts), Myra McClure (Imperial College of Science, Technology and Medicine, University of London, London, United Kingdom), and Robin Weiss (University College London, London, United Kingdom) for their generous donations of SRV-1 to SRV-5 virus strains. We thank Janet Bootman for propagation of and DNA extraction from the SRV-1 to SRV-5 virus strains used; Alan Heath for help with statistical analyses; and Neil Berry, Alison Wade-Evans, Margaret Thouless, Myra McClure, and Robin Weiss for helpful discussions.

This study was supported by a grant from the Department of Health (United Kingdom) and MRC (United Kingdom) grant G9028370.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Retrovirology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom. Phone: 44 01707 641000. Fax: 44 01707 649865. E-mail: rwilkinson{at}nibsc.ac.uk. Back


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Journal of Virology, October 2003, p. 10751-10759, Vol. 77, No. 20
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.20.10751-10759.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.




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