Isolation from African Sykes' Monkeys (Cercopithecus mitis) of a Lentivirus Related to Human and Simian Immunodeficiency Viruses

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1 JOURNAL OF VIROLOGY, Apr. 1991, p X/91/ $02.00/0 Copyright C) 1991, American Society for Microbiology Vol. 65, No. 4 Isolation from African Sykes' Monkeys (Cercopithecus mitis) of a Lentivirus Related to Human and Simian Immunodeficiency Viruses PETER EMAU,'2 HAROLD M. McCLURE,13 MOHAMED ISAHAKIA,2 JAMES G. ELSE,1 AND PATRICIA N. FULTZ.3t* Yerkes Primate Research Center' and Department of Pathology,3 Emory University, Atlanta, Georgia 30322, and Institute of Primate Research, National Museums of Kenya, Nairobi, Kenya2 Received 6 September 1990/Accepted 31 December 1990 Analysis of serum samples from 100 wild-caught or colony-born Sykes' monkeys (Cercopithecus mitis) in Kenya revealed that 59 animals had antibodies cross-reactive to human immunodeficiency virus type 2 (HIV-2) and to simian immunodeficiency viruses (SIVs). A lentivirus, designated SIVsyk, was isolated from five of six seropositive asymptomatic Sykes' monkeys, but in four cases isolation was possible only after depletion of CD8+ lymphocytes and cocultivation of the CD4+-enriched cell population with peripheral blood mononuclear cells from seronegative Sykes' monkeys. SIVsyk resembled other SIVs and HIVs morphologically, had an Mg2+-dependent reverse transcriptase enzyme, and replicated in and was cytopathic for CEMx174 and Sup-Tl cells. SIVsyk differed substantially from other SIVs, however, in that it failed to replicate in normal human, mangabey, and macaque peripheral blood mononuclear cells and serum from seropositive Sykes' monkeys immunoprecipitated env antigens from HIV-1 as well as from HIV-2, SIVsmm, and SIVagm. These data demonstrate a high prevalence of natural infection in Sykes' monkeys in Kenya with a lentivirus that appears to be unique with respect to its host range and antigenic cross-reactivity. Simian immunodeficiency viruses (SIV) and the closely related human immunodeficiency viruses (HIV-1 and HIV-2) belong to the lentivirus subfamily of retroviruses (7). Both the SIVs and HIVs are tropic for CD4+ lymphocytes and monocytes/macrophages (23-25, 29, 36) and have parallel genomic organizations (4, 8, 15) and cross-reactive antigenic epitopes. Infection of macaque monkeys with SIV isolates from various macaque species (Macaca spp.) or from sooty mangabey monkeys (Cercocebus atys) results in an immunodeficiency syndrome remarkably similar to that caused by HIV in humans (26-30, 32). SIV infection of their natural hosts, however, such as infection of sooty mangabey monkeys with SIVsmm or infection of African green monkeys (Cercopithecus aethiops) with SIVagm does not appear to cause disease (10, 17), suggesting that SIVs and their natural hosts have evolved a symbiotic relationship. The SIVs and HIVs identified to date form four genetically distinct subgroups of primate lentiviruses: SIVsmm/SIVmac/HIV-2, SIVagm, SIVmnd, and HIV-1/SIVcpz (7, 19, 41). At the molecular level, isolates of SIVmac and SIVsmm are about 80 to 90% related to each other overall, 70 to 80% related to various HIV-2 isolates, but only 40 to 50% related to HIV-1 or SIVagm or SIVmnd (4, 8, 9, 16, 41), indicating that SIVsmm, SIVmac, and HIV-2 belong to the same lentivirus subgroup. Seroepidemiologic studies have shown that macaque monkeys in their natural habitats are seronegative for SIV (34), but some seropositive sooty mangabeys have been identified in West Africa, where they are indigenous (9a, 29a). Therefore, it has been proposed that sooty mangabeys may be the natural host in the wild for the SIVsmm/SIVmac/HIV-2 subgroup (7, 16, 32). Comparisons of the genomic organization of SIVagm and SIVmnd indicate that these SIVs are distinct from all other SIVs and HIVs * Corresponding author. t Present address: Department of Microbiology, University of Alabama at Birmingham, UAB Station, BHSB Room 344, Birmingham, AL and are equidistant genetically not only from each other but also from HIV-1 and the SIVsmmISIVmac/HIV-2 subgroups (9, 40, 41). Although no known SIV isolated from a simian species is closely related to HIV-1, a lentivirus with the same genetic organization and approximately 65% overall amino acid identity to HIV-1 was isolated recently from a chimpanzee in Gabon (19, 35). It also has been proposed that several species of nonhuman primates may have harbored SIVs for a long time, in particular African green monkeys because of the extreme genetic diversity among SIVagm isolates (20, 27). Seroepidemiologic studies indicate that in addition to African green monkeys, sooty mangabeys, and mandrills, other nonhuman primates in Africa may be infected with HIV- or SIV-related lentiviruses (34). The present study documents the seroprevalence of infection with a virus cross-reactive with HIV and SIV among Sykes' monkeys housed at the Institute of Primate Research in Kenya and reports the isolation of a lentivirus, termed SIVsyk, from these monkeys. This monkey is the fifth species of African nonhuman primate from which a lentivirus has been isolated. Because of the phylogenetic relationship between Sykes' monkeys and African green monkeys, which share the same natural habitat and range, it is of special interest to determine the relatedness of SIVsyk and SIVagm. Characterization of additional SIVs may provide clues to the origins, evolution and natural history of primate lentiviruses, including HIV-1. Seroprevalence of antibodies to SIV and HIV in Sykes' monkeys. Serum samples collected over 2 years (1986 to 1988) from 100 Sykes' monkeys, both caught in the wild and born at the Institute of Primate Research, were shipped to Yerkes Primate Research Center, where they were screened for antibodies cross-reactive with HIV-2 by enzyme immunoassay (EIA; Genetic Systems). Of the 100 animals, 60 were seropositive, and 59 of these were confirmed positive by radioimmunoprecipitation (RIP) analysis with cell lines persistently infected with either SIVsmm or SIVagm, the 2135

2 2136 NOTES latter cell line provided by R. Desrosiers and M. Daniel. The RIP assays were performed as described previously (12) with human cell lines persistently infected with various viruses. Briefly, 107 cells were incubated for 18 to 20 h with [35S]cysteine and [35S]methionine (100 pici/ml) and lysed, and supernatants were clarified by centrifugation. Lysates were incubated overnight at 4 C with 10 Fl of antiserum specific for HIV or SIV and were immunoprecipitated with protein A-Sepharose beads. Proteins were disassociated from beads by boiling for 5 min in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and visualized by autoradiography after separation on 5 or 9.5% acrylamide-sds gels. For the serum samples tested by RIP analysis against both SIVsmm and SIVagm, there was 100% concordance. RIP analysis also showed that a majority of the SIV/HIV-2 seropositive Sykes' monkeys had antibodies only to the major envelope glycoprotein, with no detectable antibodies to gag antigens (data not shown). A subset of 30 serum samples were tested for crossreactivity to HIV-1 by EIA (Genetic Systems); of 16 serum samples positive for both HIV-2 and SIV, only 5 were positive in the HIV-1 EIA. The original 100 serum samples also were screened by EIA (Du Pont) for antibodies crossreactive with human T-lymphotropic virus type I (HTLV-I), which is antigenically related to simian T-lymphotropic virus type I (STLV-I); 33 of the animals (33%) were seropositive and 67 were seronegative. To assess the accuracy of the HTLV-I EIA kit, we tested 39 randomly selected serum samples by both HTLV-I EIA and RIP assay by using lysates of MT2 cells chonically infected with HTLV-I. There was discordance with only one sample, which was positive by EIA but negative by RIP, indicating that the data obtained with the HTLV-I EIA kit gave a reliable approximation of seroprevalence of infection with STLV-I. Titers in serum samples from six seropositive Sykes' monkeys were determined by HIV-1 and HIV-2 EIA (Table 1). Antibody titers ranged from 1:3,200 to 1:51,200 against HIV-2, but the highest titer against HIV-1 was only 1:400. For comparative purposes, titers in serum samples from Kenyan African green monkeys, a rhesus macaque and a sooty mangabey infected with SIVsmm, and an HIV-1- infected chimpanzee were also determined by EIA. In general, the African green monkeys had lower titers than the Sykes' monkeys against both HIV-1 and HIV-2, but this could be deceptive depending on relative amounts of specific antigens in each test. Virus isolation. Six seropositive and three seronegative animals from the Institute of Primate Research were sent to Yerkes Primate Research Center (accredited by the American Association for Accreditation of Laboratory Animal Care), where they were housed in individual cages and used as donors of peripheral blood mononuclear cells (PBMC) in virus isolation attempts. In our laboratory, previous attempts to isolate SIV by cocultivation of simian PBMC with normal human PBMC generally had been successful, so initially we attempted to isolate virus from seropositive Sykes' monkeys by this method. PBMC were obtained by separation of heparinized blood on Ficoll-sodium diatrizoate (LSM; Organon Teknika) and were maintained in RPMI 1640 to which were added 10% fetal bovine serum, glutamine, gentamicin, 8 U of recombinant interleukin-2 (Boehringer Mannheim) per ml, and DEAE-dextran (IL-2-RPMI). Approximately 107 total PBMC were cocultivated with 107 indicator cells, and cell-free supernatants were monitored periodically for Mg2+-dependent reverse transcriptase (RT) by a modified micromethod (37). Despite maintenance of the TABLE 1. Comparison of antibody titers to HIV-1 and HIV-2 of animals infected with various SIVs or HIV-1 Animal HIV-1 EIA titera J. VIROL. HIV-2 Sykes' monkeys Cm72 < 100 3,200 Cm ,800 Cm173 < ,800 Cml81 < ,800 Cm ,800 Cm ,200 African green monkeys Agm37 < 100 3,200 Agm4l < 100 6,400 Agm ,400 Agm34 < Agm33 < 100 3,200 Agm SIVsmm-infected monkeysb RIz 1,600 25,600 FHi ,800 HIV-1-infected chimpanzee C ,200 6,400 atiters are the reciprocal of the highest dilution of serum that gave a positive reading when using HIV-1 or HIV-2 EIA kits (Genetic Systems). <100, negative at a 1:100 dilution, the lowest dilution tested. b RIz is an experimentally infected rhesus macaque; FHi is a naturally infected sooty mangabey. cocultured PBMC for 10 weeks, during which time freshly stimulated human PBMC were added periodically, all of the cultures remained negative for RT activity. Ohta et al. (34) successfully obtained SIVagm isolates, which previously had been refractory to isolation, by using a cell line they developed, Molt 4-clone 8. Also, another cell line, CEM x 174, had been shown to be highly susceptible to infection, replication, and syncytia formation by most strains of SIV (9a, 18). Thus, we next attempted to isolate SIVsyk by cocultivation of Sykes' monkey PBMC with these two cell lines, which were maintained in RPMI 1640 with 10% fetal bovine serum, glutamine, and gentamicin (10%-RPMI). On the assumption that simian PBMC might support replication of another SIV more readily than PBMC of human origin would, Sykes' monkey PBMC were also cocultivated with PBMC from normal rhesus macaques. Although cocultures with rhesus macaque PBMC remained negative for RT activity, cocultures of Sykes' monkey PBMC with both cell lines contained transient low levels of RT activity after 7 weeks of culture, but virus was not detected by electron microscopy. Because CD8+ lymphocytes have been shown to suppress replication and recovery of virus from HIV-1-infected humans (39, 42, 43) and chimpanzees (12a) and from SIVinfected monkeys (39), PBMC from a seropositive Sykes' monkey, Cm173, were depleted of CD8+ cells by panning by standard procedures (38). The CD4+-enriched population then was cocultivated with lectin-stimulated PBMC from humans, rhesus macaques, or seronegative Sykes' monkeys. These last PBMC were included on the assumption that SIVsyk might have a restricted host range. Whereas the culture containing Cm173 CD4+-enriched cells and PBMC from a seronegative Sykes' monkey was positive for RT activity by day 10, neither of the cocultivations with human or rhesus PBMC as indicator cells became RT positive during 37 days of culture. The presence of virus in the

3 VOL. 65, 1991 culture with CD4+-enriched cells and PBMC from the seronegative Sykes' monkey was confirmed by electron microscopy. Mature lentivirus particles with an electron-dense rod-shaped core were observed attached to cell membranes (data not shown). Virus stocks were generated by clarification of cell debris from culture supernatants, filtration through 0.22-,um filters, and cryopreservation of multiple aliquots. Cell-free supernatants from this culture transferred infectivity to PBMC from a seronegative Sykes' monkey. This virus isolate was designated SIVsykl73 and was used as the prototype for subsequent experiments. From the results with PBMC from animal Cm173, attempts to isolate virus from the other five seropositive Sykes' monkeys included cocultivation of whole PBMC or CD4+-enriched cells with PBMC from the seronegative Sykes' monkeys. Virus replication was observed in cocultures of cells from four of the other five animals, but confirmation by electron microscopy was not done. In summary, isolation of virus was apparently successful from four of the six monkeys when using CD4+-enriched cells, which suggests that CD8+ lymphocytes in SIV-infected Sykes' monkeys also are capable of suppressing virus replication and recovery. Host range of SIVsykl73. Because SIVsyk was isolated only by coculture with PBMC from seronegative Sykes' monkeys, we evaluated the ability of cell-free SIVsykl73 to replicate in PBMC of human and nonhuman primate origin and in a variety of cell lines, which included the T-cell lines CEM, HT, Molt 4-clone 8 (obtained from M. Hayami via L. Montagnier), and Sup-Ti (obtained from J. Hoxie); the T-cell/B-cell hybrid CEM x 174 (obtained from J. Hoxie); and the promonocytic U937 line. To determine the cell tropism and host range of SIVsyk, 107 cells of the various established cell lines or lectin-stimulated PBMC from different species of monkeys (10 p.g of concanavalin A per ml), chimpanzees (10 plg of concanavalin A per ml), and humans (0.1% phytohemagglutinin P) were suspended in 2 ml of medium and inoculated with 105 cpm of RT activity from SIVsykl73. Following a 1-h adsorption, the cells were washed and resuspended at 106 cells per ml in 10%-RPMI (cell lines) or IL-2-RPMI (PBMC), and cell-free supernatants were monitored every 4 to 5 days for RT activity. Cell viability was determined by trypan blue dye exclusion. Consistent with our failure to isolate virus by cocultivation of PBMC from seropositive Sykes' monkeys with PBMC from normal humans or rhesus macaques, SIVsykl73 established a productive infection only in PBMC from seronegative Sykes' monkeys and failed to replicate in PBMC from humans, chimpanzees, mangabeys, rhesus macaques, and pig-tailed macaques. Of the cell lines tested, only CEMx174 and Sup-Ti cells supported replication of SIVsykl73 during the 48 days the cultures were monitored. Replication of SIVsykl73 appeared restricted in Sup-Ti cells compared with CEM x 174 cells because maximum virus production in Sup-Ti cells was about 10-fold lower than in CEM x 174 cells (Fig. 1). However, the virus was extremely cytopathic for both cell lines since on day 28 of culture (in the experiment shown), cell viability in the Sup-Ti and CEM x 174 cultures was only 5 and 10%, respectively. A persistent infection of CEM x 174 cells with SIVsykl73 was eventually established by periodically adding uninfected CEM x 174 cells to the culture. Identification of cross-reactive antigens. The initial EIA analysis of serum samples from Sykes' monkeys had suggested that SIVsyk was more closely related to the SIVsmm/ SIVmac/HIV-2 and SIVagm subgroups than to HIV-1. To 0. (1-) ;t C1-104 I 1o Days in culture NOTES 2137 FIG. 1. Comparative replication of SIVsykl73 in continuous human cell lines. Uninfected CEMx174 cells were added to that culture on day 28. Symbols: O, CEMx 174; V, Sup-T1; 0, U937; O, Molt 4-clone 8; A, HT. determine which specific viral proteins might contain crossreactive epitopes and to assess relative molecular weights of the major gag- and env-encoded proteins, we performed reciprocal RIP analyses by using persistently infected cell lines, including the CEMx 174 line persistently infected with SIVsykl73. Surprisingly, with serum from the Sykes' monkey with the highest HIV-2 EIA titer (Cm203), the majority of the cross-reactivity appeared to be to the envelope glycoproteins not only of HIV-2 and SIVagm but also of HIV-1 (Fig. 2A). Because the anti-sivsyk serum had low levels of antibodies to the major core protein, when compared with the amount of gag-encoded protein immunoprecipitated by the anti-hiv-1 serum, it is possible that there is some antigenic cross-reactivity between the Gag proteins of SIVsyk and the other viruses tested, but that insufficient amounts of antibodies were present to detect it. As observed by others (22), anti-hiv-1 serum immunoprecipitated the major core proteins of all other viruses but had no detectable cross-reactivity to the envelope glycoproteins. Although the apparent molecular weight of the envelope glycoprotein of SIVsyk was comparable to those of the other T-lymphotropic lentiviruses (Fig. 2B), the major gag-encoded protein appeared to be slightly larger than p24 of HIV-1 and p26 of HIV-2 and SIVagm (Fig. 2A). Of 23 SIV-positive serum samples from Sykes' monkeys that were tested by RIP analysis with HIV-1-infected cell lines, all 23 had antibodies that immunoprecipitated the HIV-1 envelope glycoprotein. In contrast, Allan et al. (1) demonstrated that serum samples reactive to gpl20env of SIVagm(gri) and SIVagm(ver) did not react with gpl20env of HIV-1. Thus, this preferential cross-reactivity of serum from SIVsyk-infected monkeys with env rather than gag antigens is anomalous among the various lentivirus subgroups. It was noteworthy that SIVsyk did not appear to infect or to replicate in human PBMC, which also makes it unique among the T-lymphotropic lentiviruses. Furthermore, the apparent inability of SIVsyk to infect PBMC from other nonhuman primates was surprising, since other SIVs and HIVs have been shown to establish infections in species other than the hosts from which they were isolated (11, 12, 17, 33). This restricted host range could be due to differences 48

4 2138 NOTES J. VIROL. A 1: a-sivsyk 2: ca-hiv-1 3: a-sivsmm 4: normal kd W 200 gp160/ gpl160 HIV- 1 p 20- B IV siv syk HIV-1 agm M kd gm_ I OW" ' p55-_. a 0 46 at 69 M * 30 p55- p24- I* 4; _ SIVsyk HIV-1 SIVagm HIV-2 M FIG. 2. RIP analysis of antigenic cross-reactivity in serum from nonhuman primates infected with different T-lymphotropic lentiviruses. Lysates of cells persistently infected with SIVsyk, HIV-1, SIVagm, or HIV-2 were immunoprecipitated with serum samples from animals infected with SIVsyk, HIV-1, or SIVsmm and analyzed on 9.5% (A) or 5% (B) SDS-gels. Molecular masses of HIV-1 Env proteins (gp160/120) and Gag proteins (p24, p55) are indicated on the left, and molecular mass standards are shown on the right. In panel B, lysates were immunoprecipitated with serum from Sykes' monkey Cm203 (lane 1), an HIV-1-infected chimpanzee (lane 3), a Kenyan African green monkey (lane 5), and a normal animal (lanes 2, 4, and 6). either in the binding site(s) on the SIVsyk glycoprotein that interacts with the CD4 cell surface molecule, which may vary among the various species, thus affecting binding affinities or capabilities, or in the putative second receptor that facilitates fusion, as has been postulated for HIV-1 and SIV (5, 18, 29). Alternatively, SIVsyk may not utilize CD4 as its primary cell surface receptor. SIVsyk could not be isolated by using multiple approaches that were successful in the initial isolations of several other primate lentiviruses; this can be explained in part by its restricted host range. This apparent restriction in growth in PBMC from other primates is not unprecedented because most SIVs do not replicate in chimpanzee PBMC. That virus was isolated from four of the six seropositive Sykes' monkeys only after enrichment for CD4+ lymphocytes suggests that CD8+ lymphocytes suppressed virus replication and/or recovery, as has been described for SIVmac-infected macaques (39) and HIV-1-infected humans (39, 42, 43) and chimpanzees (12a). CD8+ cell-mediated restriction of virus recovery also is indicative of low virus loads and is correlated with the presence of SIV-specific CD8+ cytotoxic T lymphocytes, which may be associated with prevention of disease progression in some cases (31). Although limited observations of the Sykes' monkeys at the Institute of Primate Research indicate that SIVsyk infection in this species is not associated with clinical disease, it is not known whether CD8+ cells play a role in maintenance of the asymptomatic state. Failure of a species to develop immunodeficiency disease following infection with SIV is not always associated with control of virus expression and low virus loads. Sooty mangabey monkeys naturally infected with SIVsmm have high virus loads, including high levels of cell-free virus in plasma, yet rarely develop immunodeficiency disease directly attributable to SIVsmm infection (10, 13). The more probable explanation for the mangabey's resistance to disease is the observation that natural SIVsmm isolates are not cytopathic for mangabey CD4` cells despite efficient replication of virus (9a). This also appears to be true for SIVagm infection of African green monkey PBMC (1, 17). Experiments to determine whether SIVsyk is cytopathic for CD4+ cells from Sykes' monkeys were inconclusive because, in general, SIVsyk replicated poorly, even in primary Sykes' PBMC. The data presented here show that SIV infection appears to be widespread in native populations of Sykes' monkeys, which belong to the same genus, Cercopithecus, as African green monkeys, from which an exceptionally diverse group of lentiviruses have been isolated. Although the finding of 59% seropositivity to SIV among Sykes' monkeys in Kenya is higher than the apparent prevalence (26 to 42%) of infection by SIV among Cercopithecus species in the wild (21, 34), it is in good agreement with the 57% seropositivity to SIVsmm among 138 sooty mangabeys in a captive breeding colony (10). Also in agreement was the 33% seropositivity to STLV-I in both the captive Sykes' monkeys and the mangabeys (10). Lentiviruses have now been isolated from six different primates: humans (2, 14), chimpanzees (35), macaques (3, 6, 32), mangabeys (11, 28), mandrills (40), and two Cercopithecus species, African green monkeys (1, 25, 34) and Sykes' monkeys (this report). Although Sykes' and African green monkeys share the same ranges, SIVsykl73 appears to be phenotypically distinct from SIVagm isolates, and infection of Sykes' monkeys most probably did not result from transmission across species barriers. This is supported by preliminary sequence data from a portion of the gag region of SIVsyk, which indicate that this virus is distinct from all other SIV and HIV groups, including SIVagm, and thus may be a member of a fifth primate lentivirus group (19a). We thank Becky Siegel and Ellen Lockwood for expert technical assistance; Barbara Olberding for electron microscopy; Becky Siegel and Frank Kiernan for graphics; and R. Desrosiers, M. Daniel, M. Hayami, J. Hoxie, and L. Montagnier for supplying viruses and/or cell lines.

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