Virus Subtype-Specific Features of Natural Simian Immunodeficiency Virus SIV_smm Infection in Sooty Mangabeys

Abstract

Simian immunodeficiency virus (SIV) SIV_smm naturally infects sooty mangabeys (SMs) and is the source virus of pathogenic infections with human immunodeficiency virus type 2 (HIV-2) and SIV_mac of humans and macaques, respectively. In previous studies we characterized SIV_smm diversity in naturally SIV-infected SMs and identified nine different phylogenetic subtypes whose genetic distances are similar to those reported for the different HIV-1 group M subtypes. Here we report that, within the colony of SMs housed at the Yerkes National Primate Research Center, at least four SIV_smm subtypes cocirculate, with the vast majority of animals infected with SIV_smm subtype 1, 2, or 3, resulting in the emergence of occasional recombinant forms. While SIV_smm-infected SMs show a typically nonpathogenic course of infection, we have observed that different SIV_smm subtypes are in fact associated with specific immunologic features. Notably, while subtypes 1, 2, and 3 are associated with a very benign course of infection and preservation of normal CD4⁺ T-cell counts, three out of four SMs infected with subtype 5 show a significant depletion of CD4⁺ T cells. The fact that virus replication in SMs infected with subtype 5 is similar to that in SMs infected with other SIV_smm subtypes suggests that the subtype 5-associated CD4⁺ T-cell depletion is unlikely to simply reflect higher levels of virus-mediated direct killing of CD4⁺ T-cells. Taken together, this systematic analysis of the subtype-specific features of SIV_smm infection in natural SM hosts identifies subtype-specific differences in the pathogenicity of SIV_smm infection.

The AIDS pandemic originated from zoonotic transmission to humans of simian CD4⁺ T-cell-tropic lentiviruses that infect African monkey species and are collectively defined as simian immunodeficiency viruses (SIV) (12, 24, 26). In marked contrast to human immunodeficiency virus (HIV)-infected humans, who, if untreated, almost invariably develop progressive CD4⁺ T-cell depletion and AIDS, the African monkey species naturally infected with SIV are generally spared from any signs of disease (reviewed in references 28, 33, 54, and 64). Conversely, experimental SIV infection of nonnatural host Asian macaque species causes symptoms similar to those described for AIDS patients (simian AIDS) (reviewed in reference 32). At present, it is still unclear why SIV infection is relatively nonpathogenic in natural host monkey species but induces immunodeficiency in nonnatural or recent hosts (including humans). A better understanding of the mechanisms underlying the lack of disease in natural hosts for SIV infection will likely provide important clues as to the pathogenesis of AIDS in HIV-infected individuals (58, 59).

Among the natural hosts for SIV infection, the sooty mangabeys (SMs) (Cercocebus atys) are particularly relevant for two reasons. First, SIV_smm, the virus infecting SMs, is the root cause of the HIV type 2 (HIV-2) epidemic in humans (12, 25). Second, SIV_smm was used to generate the various rhesus macaque (Rh)-adapted SIV_mac/SIV_smm viruses (i.e., SIV_mac239 and SIV_smm543-3) that are commonly used for studies of AIDS pathogenesis and vaccines (reviewed in reference 32). SIV_smm-infected SMs typically maintain normal CD4⁺ T-cell counts and do not develop AIDS despite intensive virus replication, with levels of plasma viremia that are as high as, or even higher than, those observed in HIV-infected individuals (11, 51, 56). Therefore, unlike both HIV-infected individuals and experimentally SIV-infected Rhs, in which high levels of virus replication predict faster disease progression (31, 35, 39, 40, 47, 66), in SMs the levels of SIV_smm viral loads (VLs) do not seem to have a predictive value for the outcome of infection (11, 51, 56).

Natural SIV infection of SMs is characterized by near-normal CD4⁺ T-cell counts in both blood and lymph nodes, normal T-cell turnover, preserved bone marrow and thymic function, and absence of the generalized immune activation and bystander T-cell apoptosis that are typical of HIV-infected individuals (11, 56, 60). Similarly, no signs of disease progression and relatively low levels of immune activation were observed in SMs experimentally infected with uncloned SIV_smm or molecularly cloned SIV_mac239 (34, 55). Taken together, these results led to the hypothesis that the attenuated immune activation in SIV-infected SMs is a mechanism that favors the preservation of CD4⁺ T-cell homeostasis and, thus, the nonpathogenic state of this chronic infection.

Interestingly, the SIV_smm cross-species transmission to both humans and macaques has specific epidemiological and pathogenic features, with some of the strains showing better fitness in terms of transmission to and replication in the new hosts. Only groups A and B of HIV-2 resulted in epidemics (19, 25), whereas the remaining HIV-2 groups act as epidemiological dead ends in humans, with lower pathogenicity and no documented human-to-human transmission (12, 25, 67). Similarly, SIV_mac strains clearly have higher pathogenic potential than other cross-species-transmitted SIV_smm strains both in Rhs and in other macaque species, such as pig-tailed macaques or stump-tailed macaques (32, 42, 46). We and others suggested that a key factor beyond these differences in pathogenicity of cross-species-transmitted SIV_smm in the macaque host may be the serial passage in vitro and/or in vivo (4, 6, 8, 10, 30). However, the alternative hypothesis that different SIV_smm strains show intrinsic differences in pathogenicity cannot be dismissed.

The recent discovery of a significant SIV_smm diversity in naturally infected SIVs from primate centers in the United States (3) offered us the possibility to directly test this later hypothesis. In this study we have characterized the SIV_smm diversity within the large colony of naturally SIV-infected SMs housed at the Yerkes National Primate Research Center (YNPRC) and correlated the prognostic markers of disease progression (levels of CD4⁺ T cells, VLs, and T-cell activation) with SIV_smm lineage. We have also performed a longitudinal retrospective study of the dynamics of virus replication in naturally SIV-infected SMs from the Tulane National Primate Research Center (TNPRC) over a 20-year period. While these studies failed to reveal any significant differences between the known SIV_smm lineages in terms of virus replication or progression to AIDS, a significant association between subtype 5 infection and CD4⁺ T-cell depletion was observed.

MATERIALS AND METHODS

Animals.

The cross-sectional study included 110 naturally SIV-infected SMs from YNPRC and 29 from TNPRC. At least one blood sample was collected from all animals. The retrospective study involved serum samples collected over a 20-year period from 20 naturally infected SMs from the TNPRC, which were infected with six different subtypes as follows: eight with subtype 1 (AO24, EO38, EO39, EO41, GO79, M923, M927, and M939), three with subtype 2 (D177, GO80, and M946), three with subtype 3 (AO23, F102, and M942), three with subtype 4 (G930, G931, and G932), two with subtype 5 (D175 and FO98), and one with subtype 6 (D215). At least five serum samples and up to 18 serum samples were available from each SM included in this retrospective study. Moreover, for the SMs in the TNPRC colony, a prospective study of virus replication in plasma samples was carried out for 6 years to observe the dynamics of SIV_smm VLs. All SMs and Rhs housed at the YNPRC and TNPRC were maintained in accordance with NIH guidelines (44).

Nucleic acid extractions.

RNA was extracted from plasma and serum of all SIV_smm-infected SMs from TNPRC and YNPRC using the QIAamp viral RNA kit (QIAGEN, Valencia, CA), as described previously (3).

PCR and sequencing.

Nested PCR was performed to obtain amplified fragments from the gag, env, and pol regions, as described previously (3). A 793-bp gag fragment was obtained by a nested PCR protocol using GagA/GagB and GagC/GagF primers, as described previously (25). Alternatively, primers GF1/GR1 and GF2A/GR3 (13) were used in a nested PCR to generate a 909-bp fragment in the gag region. These two fragments completely overlapped. Nested primers were used for sequencing. A 438-bp fragment in the gp36 env region was obtained using a nested PCR protocol with the primers EF4/ER1 and EF5A/ER2A (12). A 602-bp pol integrase fragment was obtained using a slight variation of previously described primers used to amplify divergent SIVs (14-17). Polis4B (5′-CCA GCH CAY AAA GGW ATA GGW GGA AA-3′) and PolORB (5′-ACT GCH CCT TCH CCT TTC CA-3′) were used in the first round of amplification, and Polis4B was again used in a seminested reaction with Unipol2B (5′-CCC CTA TTC CTC CCY TTC TTT TAA-3′). PCR conditions were as previously described (3).

PCR products were purified using the QIAquick gel extraction kit or the PCR purification kit (QIAGEN, Valencia, CA) and sequenced by using direct sequencing and dye terminator methodologies (ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq FS DNA polymerase [Applied Biosystems, Foster City, CA]) on an automated sequencer (ABI 373, stretch model; Applied Biosystems). Sequencing was performed using the inner primers of each reaction.

Phylogenetic analysis.

The gag, pol, and env nucleotide sequence alignments were obtained from the Los Alamos National Laboratory HIV Sequence Database (http://hiv-web.lanl.gov). Newly derived SIV_smm sequences were aligned using the CLUSTALW (63) profile alignment option. The resulting alignments were adjusted manually where necessary. Regions of ambiguous alignment and all gap-containing sites were excluded.

Phylogenetic trees were inferred from the nucleotide sequence alignments by the neighbor-joining method (52) using PAUP* (62) and based upon the HKY85 model of nucleotide substitution (29). The reliability of branching order was assessed by performing 1,000 bootstrap replicates, again utilizing neighbor joining and the HKY85 model. Phylogenetic trees were also inferred by maximum likelihood using PAUP* with models inferred from the alignment using Modeltest (50). The neighbor-joining tree topology was used as the starting tree in a heuristic search employing tree-bisection-reconnection branch swapping.

Determination of plasma viral RNA.

For the cross-sectional study, SIV_smm RNA quantitation was performed by real-time PCR as follows. RNA was extracted and reverse transcribed as described previously (55). Real-time PCR was performed by amplification of 20 μl cDNA in a 50-μl reaction mixture containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 4 mM MgCl₂, 0.2 μM forward primer, 0.3 μM reverse primer, 0.1 μM probe, and 5 U of AmpliTaq Gold DNA polymerase (reagents from Applied Biosystems, Foster City, CA). Primer and probe sequences were targeted to the 5′ untranslated region of the SIV_smm genome: forward primer, 5′-GGCAGG AAAATCCCTAGCAG-3′; reverse primer, 5′-GCCCTTACTGCCTTCACTCA-3′; probe, 5′-(6-carboxyfluorescein)-AGTCCCTGTTCRGGCGCCAA-(6-carboxyltetramethylrhodamine). Amplicon accumulation was monitored with an ABI PRISM 7700 sequence detection system (Applied Biosystems) with the following cycling conditions: 50°C for 2 min, 95°C for 10 min, 40 cycles at 93°C for 30 s, and 59.5°C for 1 min. RNA copy number was determined by comparison to an external standard curve consisting of in vitro transcripts representing bases 216 to 2106 of the SIV_mac239 genome.

For the retrospective study of SIV_smm virus replication dynamics in naturally infected SMs, VL was measured on serum stored at −70°C and thawed only once before testing. For the prospective study, VLs were quantified on fresh plasma samples using the branched DNA (bDNA) assay (Bayer Reference Testing Laboratory, Emeryville, CA) that we have previously reported to properly detect different SIV_smm lineages (2, 3).

Lymphocyte studies and flow cytometry.

Whole blood was stained using a whole-blood lysis technique as previously described (48). Monoclonal antibodies for flow cytometry which were originally designed to detect human molecules have been shown by us and others to be cross-reactive with SMs (55, 56, 65). The antibodies used were anti-CD4-phycoerythrin (clone SK3), anti-CD4-allophycocyanin (clone SK3), and anti-CD8-allophycocyanin (clone SK1) (all from Becton Dickinson, San Jose, CA) and anti-CD3-phycoerythrin (clone SP34-2) (from BD Pharmingen, San Diego, CA). Flow cytometric acquisition and analysis of samples were performed on at least 100,000 events on a FACSCalibur flow cytometer driven by the CellQuest software package (Becton Dickinson). Analysis of the acquired data was performed using FlowJo software (Tree Star, Inc., Ashland, OR). Cell blood counts were performed for each animal and each time point and were used to determine the absolute numbers of CD4⁺ T cells.

Statistical analyses.

VL data and CD4⁺ T-cell levels were displayed using the box-and-whisker plot method. In all figures displaying box plots, the box extends from the 25th percentile to the 75th percentile, with a horizontal line at the median, and the whiskers extend down to the 10th percentile and up to the 90th percentile. Each outlier is shown as an individual point outside the plots. SigmaPlot 5.0 software (SPSS, Inc., Richmond, CA) was used for box plot construction.

The Kruskal-Wallis one-way analysis of variance with the post hoc Tukey test was used to analyze the statistically significant differences in VLs and CD4⁺ T-cell counts between SMs infected with different SIV_smm subtypes or uninfected SMs, with P < 0.05 being considered significant.

Nucleotide sequence accession numbers.

The nucleotide sequences of the gag, pol, and env sequences from SIV_smm-infected SMs were deposited in GenBank (accession numbers EF569684 to EF569963).

RESULTS

Four distinct SIV_smm subtypes cocirculate at the YNPRC.

This extensive study of SIV_smm diversity in the colony of SMs housed at the YNPRC was prompted by our recent finding that SIV_smm diversity at the TNPRC is much higher than was initially believed, with six different phylogenetic lineages resulting from independent introductions of SIV_smm and now cocirculating at the TNPRC (3). Since part of the TNPRC colony was established with animals from YNPRC, in a previous study we investigated the SIV_smm diversity in the YNPRC colony and confirmed the presence of subtypes 1, 2, 3, and 5 in a small group of samples (3). To better characterize the extent of virus diversity and to evaluate its biological consequences, we have now extended this investigation to the entire colony of SIV_smm-infected SMs from YNPRC. To the best of our knowledge, this assessment of SIV diversity is the largest ever performed in a natural host species. We included in our study plasma samples from 110 SIV_smm-infected SMs from the YNPRC colony. At least one fragment was obtained in 105 SIV_smm-infected SMs. For the remaining ones, low VL levels precluded a positive PCR amplification, in spite of repeated attempts. Consistent with our previous report (3), the sequence analysis of SIV_smm samples identified subtypes 1, 2, 3, and 5 (Table 1; Fig. 1a to c). Similar to the distribution at the TNPRC, the distribution of SIV_smm subtypes at the YNPRC is uneven, with subtype 1 strains being the major viral form at YNPRC with 51% (54/105). Subtypes 2 and 3 have relatively similar prevalences, accounting for 20% (21/105) and 23% (24/105) of cases, respectively. Subtype 5 was detected in only three monkeys (4%). Interestingly, three SMs at the YNPRC showed recombinant profile patterns, as follows: FDv (2^gag/3^pol/3^pol), FRc (1^gag/3^env), and FPc (1^gag/1^pol/2^env).

TABLE 1.

SIV_smm subtype distribution in different age groups of SIV_smm-infected SMs from YNPRC

Age (yr)	No. (%) of SMs
	Total	With subtype:				Recombinant
		1	2	3	5
<10	19 (18)	9 (48)	4 (21)	5 (26)		1 (5)
11-15	50 (48)	26 (52)	10 (20)	13 (26)	1 (2)
16-20	22 (21)	12 (54)	7 (32)	2 (9)	1 (5)
21-25	11 (10)	6 (55)		4 (36)	1 (9)
>25	3 (3)	1 (33)				2 (66)
Total	105 (100)	54 (51)	21 (20)	24 (23)	3 (3)	3 (3)

FIG. 1.

SIV_smm diversity in SMs from YNPRC. Subtypes are clusters of SIVs that are highly related and branch together. SIV_smm strains belonging to different subtypes are color coded: red, subtype 1; violet, subtype 2; turquoise, subtype 3; blue, subtype 4; orange, subtype 5; light green, subtype 6. Reference SIV_smm strains and strains from wild-caught SMs are shown in black, whereas HIV-2 strains are shown in light gray. The trees are based on gag (500-bp) (a), pol (592-bp) (b), and env (405-bp) (c) fragments after gap-containing sites have been removed. The phylogenetic trees were estimated by the neighbor-joining method on amino acid sequences. The reliability was estimated from 1,000 bootstrap replicates; only relevant bootstrap values are shown. The scale bar indicates amino acid replacements per site. Strain nomenclature includes the assigned lineage, the primate center of origin, the year of strain collection, and the monkey identification.

We then compared the relative prevalences of different SIV_smm subtypes among age groups (Table 1). We did not observe any significant correlation for the four subtypes cocirculating at the YNPRC, suggesting that there is no significant difference in strain transmissibility over time. Interestingly, the recombinant strains are present only in a young animal and in two very old animals. This result is not entirely unexpected, as the chances of superinfection and recombination may increase with the duration of infection.

Similar levels of in vivo viral replication between SIV_smm strains belonging to different subtypes.

We then pooled data relative to all SIV_smm-infected SMs for which sequence data were available (i.e., both YNPRC and TNPRC) and compared the in vivo viral replication levels for the SIV_smm strains belonging to different subtypes (Fig. 2). The cross-sectional analysis showed a slight, albeit statistically significant, higher level of viral replication for subtype 1 strains (average, 203,162 SIV_smm RNA copies/ml; limits, 3,730 to 1,909,700 SIV_smm RNA copies/ml) than for subtype 2 strains (average, 109,858 SIV_smm RNA copies/ml; limits, 6,980 to 678,000 SIV_smm RNA copies/ml; P = 0.025), subtype 3 strains (average, 141,884 SIV_smm RNA copies/ml; limits, 460 to 1,010,000 SIV_smm RNA copies/ml; P = 0.025), and subtype 5 strains (average, 72,901 SIV_smm RNA copies/ml; limits, 301 to 187,000 SIV_smm RNA copies/ml; P = 0.0322). No statistically significant difference was observed for the recombinant strains (average, 185,613 SIV_smm RNA copies/ml; limits, 1,330 to 453,000 SIV_smm RNA copies/ml; P > 0.5) (Fig. 2). The slightly higher levels of VL observed in subtype 1-infected SMs might suggest either that subtype 1 strains are associated with replicative fitness in vivo (thus also explaining the higher prevalence of this subtype in both colonies) or that the VL quantification assay used is more sensitive for subtype 1 strains, i.e., the most numerous known at the time of assay design and optimization.

FIG. 2.

Cross-sectional analysis of plasma VL levels in 137 SMs infected with different SIV_smm subtypes. Subtypes are color coded as follows: red, subtype 1; violet, subtype 2; turquoise, subtype 3; blue, subtype 4; orange, subtype 5; light green, subtype 6. VL was quantified by bDNA (detection limit, 125 copies/ml) and real-time PCR (detection limit, 100 copies/ml). Box-and-whisker plots show the boxes that extend from the 25th percentile to the 75th percentile, with a horizontal line at the median, and the whiskers extend down to the 10th percentile and up to the 90th percentile. Each outlier is shown as an individual point outside the plots.

To further address these issues, we performed a retrospective study of the dynamics of VL in a subset of SIV_smm-infected SMs housed at TNPRC by using stored serum samples that were collected over a 20-year period. Due to the nature of samples, the VL levels quantified on sera were lower than those obtained by quantification in plasma. Importantly, this retrospective study failed to reveal any statistically significant difference in viral replication between subtype 1 (Fig. 3a) strains and non-subtype 1 strains (Fig. 3b). This analysis also revealed that the set point levels of virus replication are very stable over long periods of time in chronically SIV_smm-infected SMs, thus identifying another notable difference between nonpathogenic SIV infection of natural hosts and HIV infection of humans, where virus replication tends to increase during the course of infection.

FIG. 3.

Retrospective analysis of the dynamics of VL in SMs infected with different SIV_smm subtypes over a 20-year period showed no statistically significant difference in viral replication between subtype 1 (a) and non-subtype 1 (b) infections. Subtypes are color coded as follows: red, subtype 1; violet, subtype 2; turquoise, subtype 3; blue, subtype 4; orange, subtype 5; light green, subtype 6. Black lines show the dynamics of VLs in an SM that progressed to AIDS (36). VL was quantified by bDNA (detection limit, 125 copies/ml) on serum samples stored at −80°C.

As quantification of SIV_smm VL in serum samples may not accurately reflect the actual level of in vivo viral replication, we also collected and analyzed a set of longitudinal data in SIV_smm-infected SMs from the TNPRC in which the level of viral replication was measured in freshly collected plasma samples over a 6-year period. As shown in Fig. 4, VL levels quantified on fresh plasma exhibited remarkably well-conserved patterns and confirmed the lack of any significant difference between the different SIV_smm subtypes with respect to trends of viral replication over time.

FIG. 4.

Prospective analysis of the dynamics of viral replication of different SIV_smm subtypes over 6 years. No statistical difference in VLs between subtype 1 (red), subtype 2 (violet); subtype 3 (turquoise), subtype 4 (blue), subtype 5 (orange), and subtype 6 (light green) was seen. VL was quantified by bDNA (detection limit, 125 copies/ml).

SIV_smm subtype 5 infection is associated with significant in vivo CD4⁺ T-cell depletion.

To then determine whether infection with different SIV_smm subtypes is associated with specific immunological features, we next compared the levels of CD4⁺ T cells in the SMs infected with different SIV_smm phylogenetic subtypes to those measured in non-SIV_smm-infected SMs. As shown in Fig. 5, no statistically significant difference was observed between uninfected SMs (average, 1,061 ± 55 CD4⁺ T cells/μl; limits, 453 to 1,640 CD4⁺ T cells/μl) and those infected with subtype 1 (average, 1,016 ± 77 CD4⁺ T cells/μl; limits, 3 to 2,717 CD4⁺ T cells/μl), subtype 2 (average, 982 ± 74 CD4⁺ T cells/μl; limits, 357 to 1,854 CD4⁺ T cells/μl), and subtype 3 (average, 1,062 ± 95 CD4⁺ T cells/μl; limits, 200 to 2,273 CD4⁺ T cells/μl). Interestingly, we found a significant difference in the CD4⁺ T-cell counts between uninfected SMs and those infected with subtype 5 strains (average, 407 ± 210 CD4⁺ T cells/μl; limits, 24 to 890 CD4⁺ T cells/μl) (P < 0.02). Similarly, when the CD4⁺ T-cell counts of subtype 5-infected SMs were compared to those of SMs infected with subtype 1, subtype 2, and subtype 3, we observed statistically significant differences (P < 0.02). Consistent with this clear trend towards lower CD4⁺ T-cell counts in subtype 5-infected SMs is the intriguing observation that three out of four (75%) of these animals exhibited a CD4⁺ T-cell count of <500/μl, while only 11.2% of the non-subtype-5-infected SMs showed a decline of CD4⁺ T cells below 500/μl. Note that one subtype 5-infected SM that harbored the lowest VL (<500 copies/ml) showed the most severe CD4⁺ T-cell depletion, with only three CD4⁺ T cells/μl being detected in this animal. As none of the subtype 5-infected SMs with low CD4⁺ T-cell counts have hitherto shown any signs of illness, the actual clinical significance of the CD4⁺ T-cell depletion associated with SIV_smm subtype 5 infection is still unknown.

FIG. 5.

Cross-sectional analysis of CD4⁺ T-cell levels in SMs infected with different SIV_smm subtypes. Subtypes are color coded: subtype 1, red; subtype 2, violet; subtype 3, turquoise; subtype 4, blue; subtype 5, orange; subtype 6, light green. Statistically significant lower CD4⁺ T-cell counts were observed in subtype 5-infected SMs. Box-and-whisker plots show the boxes that extend from the 25th percentile to the 75th percentile, with a horizontal line at the median, and the whiskers extend down to the 10th percentile and up to the 90th percentile. Each outlier is shown as an individual point outside the plots.

DISCUSSION

In this study we have characterized the SIV_smm diversity in the YNPRC SM colony. Based on a large set of data, we have investigated if viral diversity has any significant impact on SIV_smm pathogenesis in its natural hosts, the SMs. We did not observe any significant difference in virulence between different SIV_smm subtypes in regard to viral replication or disease progression. However, we found a significant correlation between infection with SIV_smm subtype 5 and lower CD4⁺ T-cell counts, suggesting that at least this virus subtype may influence the immunological course of infection in the natural SIV_smm hosts.

There are two major reasons to study SIV_smm diversity in naturally infected SMs. The first reason is that the magnitude of diversity of different SIV_smm subtypes is similar to that of HIV-1 group M subtypes (3), thus indicating that SIV_smm subtypes can be used as a model of the effect of virus diversity in the setting of studies of candidate HIV/AIDS vaccines. To this end it should be remembered that the problem of viral diversity is arguably the most formidable obstacle for the development of an effective HIV vaccine (22, 27). The second reason is to characterize the virus and host factors underlying the lack of pathogenicity of SIV_smm in SMs. As recent studies (53) indicated that viral factors, such as nef, may be involved in determining the nonpathogenic phenotype of natural SIV infection, an extensive investigation of the relationship between different SIV_smm strains and the outcome of infection may help elucidate the role of virus-related factors in SMs.

In humans, the study of HIV subtype distribution allowed worldwide viral spread to be monitored: numerous studies have shown that subtype prevalence can vary significantly from country to country and within population groups and can change with time (7, 49). Numerous studies have reported that in HIV-1-infected patients, viral diversity potentially impacts the pathogenesis of infection, although such data were not always confirmed (1, 5, 7, 18, 20, 21, 57). Finally, we and others have shown that HIV diversity may have a significant impact on the efficacy of currently available diagnostic and monitoring tests as well as of antiretrovirals (5, 7, 18, 37, 38, 41, 49, 61).

In this study, we used a very large set of samples to systematically investigate the impact of viral diversity on SIV pathogenesis in its natural SM host. Our present data confirmed a preliminary study (3) in demonstrating the cocirculation of four different SIV_smm clusters at the YNPRC. We found that subtype 1, which includes reference strains B670, H9/PBj, and H4/660/543-3 (23, 30, 32, 43, 45), is the most prevalent, being responsible for approximately half of the cases (Table 1). Subtypes 2 and 3 are each found in approximately 20% of animals, thus accounting for the majority of the remaining samples. Not unexpectedly, the cocirculation of different viral subtypes resulted in the emergence of recombinant strains that we found in three SIV_smm-infected SMs. It should be noted that the current analysis relies on limited genomic information (i.e., amplification of relatively short fragments of different genomic regions); therefore, the actual number of recombinants could have been underestimated because of strains harboring the recombinant sequences in regions that were not analyzed.

To assess the circulation of SIV_smm subtypes over time, we first used the age of infected SMs as a surrogate of the time of infection; however, we found no significant differences in the relative prevalences of the different subtypes in animals of specific age groups, suggesting that the transmission of different SIV_smm subtypes was evenly distributed over time. In this perspective, the higher prevalence of recombinant strains in older animals is not surprising, as the probability of superinfection and recombination increases with the length of infection (and thus with age). The infection of a young animal with a recombinant strain is probably the result of the transmission of the recombinant form and may indicate that recombinant forms started to circulate in the colony, similar to what was described elsewhere for HIV-1 strains isolated from regions in which different subtypes cocirculate (9).

Our large cross-sectional and longitudinal analyses failed to reveal any significant difference in the in vivo replication of SIV_smm strains belonging to different subtypes. One strength of this study was the availability of a unique set of longitudinal samples that allowed us to perform long-term (i.e., up to 20 years) follow-up of the virus replication dynamics. We thus determined that, although the cross-sectional study suggested that the VLs in subtype 1-infected SMs are higher than in those infected with non-subtype 1 strains, both retrospective and prospective longitudinal studies failed to identify any subtype 1-specific features and showed similar levels of viral replication for the different strains belonging to subtypes 1 to 6 (Fig. 3 and 4). Interestingly, the retrospective analysis of VLs over a 20-year period revealed that the set point VL was peculiarly high in the SM that progressed to AIDS after an incubation period of 18 years (36), suggesting that a threshold of viral replication might predict disease progression in those rare cases of AIDS in natural hosts of SIVs.

Intriguingly, we found that SIV_smm subtype 5-infected SMs consistently displayed lower levels of CD4⁺ T cells than did SMs infected with other SIV_smm subtypes or uninfected animals. A recent survey of the majority of SIV-infected SMs housed at the YNPRC colony revealed that while 85 to 90% of these animals exhibit the healthy levels of CD4⁺ T cells previously described (56), a subset (10 to 15%) of SIV-infected SMs showed significant CD4⁺ T-cell depletion (60). Irrespective of these low CD4⁺ T-cell counts, these animals are still asymptomatic, although the recent description of a case of AIDS in a CD4-depleted, SIV-infected SM at the TNPRC (36) suggests that this asymptomatic phase may be temporary. In any event, the discovery of these “CD4-low” SMs identified a group of animals whose infection may represent an intermediate step between the nonprogressing infection of most SIV-infected SMs and the typical disease progression of HIV-infected individuals or SIV-infected Rhs (60). In this study we showed that, although “CD4-low” SMs were found among animals infected with diverse SIV_smm subtypes (suggesting that the CD4⁺ T-cell depletion is due to specific host factors), there was a strong association between infection with subtype 5 of SIV_smm and low CD4⁺ T-cell counts, with three out of four subtype 5-infected SMs showing levels of CD4⁺ T cells below 500/μl. These low CD4⁺ T-cell counts were not due to a down-modulation of the CD4 molecule from the cell surface, as no CD3⁺ cells with intracellular expression of CD4 were found (data not shown). The significance of this “CD4-low” phenotype is unknown. It should be noted, however, that in a group of experimentally infected SMs, a severe CD4⁺ T-cell depletion occurred in a subset of SMs, as a consequence of an expanded coreceptor usage, even though these animals remained asymptomatic after >5 years of follow-up (J. M. Milush, J. D. Reeves, S. Gordon, D. Zhou, A. Muthukumar, D. Kosub, E. Chacko, L. Giavedoni, C. Ibegbu, K. Stefano-Cole, J. Miamidian, M. Paiardini, A. P. Barry, S. Staprans, G. Silvestri, and D. L. Sodora, submitted for publication). Understanding why SIV-infected SMs only rarely develop CD4⁺ T-cell depletion and, perhaps even more puzzlingly, tend to avoid progression to AIDS even when their CD4⁺ T-cell counts reach AIDS-defining levels could be helpful to delineate the mechanisms underlying the highly variable time to AIDS in CD4⁺ T-cell-depleted HIV-infected humans.

In conclusion, this study of the subtype-specific features of SIV_smm infection in natural SM hosts identified, for the first time, subtype-specific differences in the pathogenicity of SIV_smm infection. It is hoped that the identification of these differences may provide guidance for further studies of HIV pathogenesis and vaccines where pathogenic SIV_smm infection of nonnatural hosts (i.e., Asian macaques) is used as an experimental model.