Isolation and Partial Characterization of a Lentivirus from Talapoin Monkeys (Myopithecus Talapoin)

Virology 260, 116–124 (1999) Article ID viro.1999.9794, available online at http://www.idealibrary.com on

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provided by Elsevier - Publisher Connector Isolation and Partial Characterization of a Lentivirus from Talapoin Monkeys (Myopithecus talapoin)

Albert D. M. E. Osterhaus,*,1 Niels Pedersen,† Geert van Amerongen,‡ Maarten T. Frankenhuis,§ Marta Marthas,† Elizabeth Reay,† Timothy M. Rose,¶,2 Joko Pamungkas,ʈ,3 and Marnix L. Bosch*,ʈ,4

*Laboratory of Immunobiology, National Institute for Public Health and Environmental Protection, Bilthoven, The Netherlands; †California Regional Primate Research Center, Davis, California 95616-8542; ‡Central Animal Laboratory, National Institute for Public Health and Environmental Protection, Bilthoven, The Netherlands; §Artis Zoo, Amsterdam, The Netherlands; ¶Pathogenesis Corporation, Seattle, Washington 98119; and ʈRegional Primate Research Center and Department of Pathobiology, University of Washington, Seattle, Washington 98195 Received February 8, 1999; returned to author for revision March 29, 1999; accepted May 5, 1999

We have identified a novel lentivirus prevalent in talapoin monkeys (Myopithecus talapoin), extending previous observations of human immunodeficiency virus-1 cross-reactive antibodies in the serum of these monkeys. We obtained a virus isolate from one of three seropositive monkeys initially available to us. The virus was tentatively named simian immunodeficiency virus from talapoin monkeys (SIVtal). Despite the difficulty of isolating this virus, it was readily passed between monkeys in captivity through unknown routes of transmission. The virus could be propagated for short terms in peripheral blood mononuclear cells of talapoin monkeys but not in human peripheral blood mononuclear cells or human T cell lines. The propagated virus was used to infect a naive talapoin monkey, four rhesus macaques (M. mulatta), and two cynomolgus macaques (M. fascicularis). All animals seroconverted and virus could be reisolated during a short period after experimental infection. A survey of SIVtal-infected captive talapoin monkeys revealed a relative decrease in CD4ϩ cell numbers in chronically (Ͼ2 years) infected animals. No other signs of immunodeficiency were observed in any of the infected animals. PCR amplification followed by DNA sequencing of two fragments of the polymerase gene revealed that SIVtal is different from the presently known lentiviruses and perhaps most related to the SIV from Sykes monkeys. © 1999 Academic Press

INTRODUCTION cases resulted in the induction of acquired immune de- ficiency syndrome-like symptoms associated with CD4 Simian immunodeficiency (SIV) viruses have been depletion in the newly infected animals. One example is identified in a number of Old World monkey species like the transmission of the SIVsm virus from sooty manga- sooty mangabeys (Cercocebus atys) (Fultz et al., 1990, beys to rhesus macaques, resulting in disease and death 1986; Gardner, 1989; Hirsch et al., 1989; Kanki et al., in these animals (Chalifoux et al., 1987; Letvin et al., 1985; 1987), four subspecies of African green monkeys (Cer- Lewis et al., 1992). Likewise, the SIVsm strain is believed copithecus aethiops) [(Allan et al., 1990; Daniel et al., 1988; Fomsgaard et al., 1990; Fukasawa et al., 1988; to be the precursor to human immunodeficiency virus Hirsch et al., 1993b; Kanki et al., 1985; Kraus et al., 1989; (HIV)-2 found in humans, through zoonotic transmission Ohta et al., 1988), and mandrills (Papio sphinx) (Tsujimoto (Gao et al., 1992). Similarly, HIV-1 is thought to be the et al., 1988, 1989) and recently from Sykes monkeys result of one or multiple zoonotic transmissions of a (Cercopithecus mitis albogularis) (Hirsch et al., 1993a), lentivirus occurring in African primates. Based on ge- sabaeus monkeys (Jin et al., 1994), red-capped manga- netic similarities lentiviruses isolated from chimpanzees beys (Chen et al., 1998; Georges-Courbot et al., 1998), a (SIVcpz) are the closest relatives to HIV-1 (Gao et al., drill monkey (Mandrillus leucophaeus) (Clewley et al., 1999; Huet et al., 1990; Janssens et al., 1994; Peeters et 1998), and from a L’Hoest monkey (Hirsch et al., 1999). al., 1992; Sakuragi et al., 1992; Vanden Haesevelde et al., Transfer of such viruses to other species has in some 1996). These data strongly suggest that HIV-1 infection of humans arose through cross-species transmission of SIVcpz from chimpanzees into humans (Gao et al., 1999).

1 Present address: Laboratory of Virology, Erasmus University, Rot- Antibodies cross-reactive to HIV-1 antigens, particu- terdam, The Netherlands. larly the major gag proteins, have been detected in a 2 Present address: Department of Pathobiology, University of Wash- number of African primates, suggesting widespread in- ington, Seattle, WA 98195. fection with HIV-1-related lentiviruses (Hayami et al., 3 Present address: Primate Research Center and School of Veteri- 1994; 1985; Lowenstine et al., 1986; Ohta et al., 1988). Of nary Medicine, Institut Pertanian Bogor, Bogor, Indonesia. 4 To whom reprint requests should be addressed. Fax: (206) 543- particular interest were the talapoin monkeys (Miopithe- 3873. E-mail: [email protected]. cus talapoin), the smallest species of Old World monkeys

0042-6822/99 $30.00 Copyright © 1999 by Academic Press 116 All rights of reproduction in any form reserved. LENTIVIRUS FROM TALAPOIN MONKEYS 117 living in west-central Africa, whose sera contained anti- Under these conditions, noticeable cell replication bodies cross-reacting to the envelope gene products in started around 6 weeks postcocultivation. The resulting addition to the major gag proteins (Lowenstine et al., cell population (designated TL-4 cells) grew in culture for 1986). The envelope gene is the most variable of lentivi- extended periods of time (weeks) in the presence of ral genes and cross-reactive antibodies to its gene prod- human IL-2, but as in the cultured PBMCs, there was a ucts would suggest a relatively close genetic relation- strong tendency for overgrowth by the CD8ϩ cells. These ship between the viruses. Attempts to isolate virus from cocultivated cells produced HTLV-1, as demonstrated by seropositive talapoin monkeys were unsuccessful. Anal- RT-PCR on culture supernatant (data not shown). The ysis of mitochondrial 12S rRNA sequences place these CD4 fraction of these cells could be infected with cell- monkeys on a separate branch of the primate phyloge- free virus isolated from talapoin monkey PBMCs (see netic tree rather than on the same branch as the Cerco- below) and produced relatively large amounts of virus, pithecus monkeys with which it is commonly classified which was subsequently used for some of the in vivo (van der Kuyl et al., 1995). They live in large groups in the infection experiments. rainforests of Gabon, where they are commensal with humans and are known to raid crops (Napier and Napier, Virus isolation 1985). Their close interactions with humans would make PBMCs from three talapoin monkeys were isolated a lentivirus that infects talapoin monkeys a candidate for and cultured in complete media supplemented with 105 cross-species transmission into humans. irradiated CEMx174 cells. The culture supernatants were In this study, we identified three HIV-1 seropositive monitored twice every week for HIV-1 p24 cross-reactive talapoin monkeys in a zoo in the Netherlands, in concor- antigen. One of the cultures produced detectable antigen dance with previous observations in American zoos. after 2 weeks of culture. Antigen production persisted for Monitoring the colony in the Netherlands provided evi- up to 6 weeks and then gradually decreased to nonde- dence for active transmission of virus because more tectable levels. Samples of these cultures were viably animals became positive over time. This sequential se- frozen at weekly intervals. The addition of fresh talapoin roconversion of previously seronegative monkeys that monkey PBMCs, or of human primary cells or cell lines, were group housed provides evidence for horizontal did not increase antigen production or extend the period transmission of SIVtal. Such active transmission may of antigen positivity. Cell-free passage of the putative have enhanced our chances to isolate virus from these virus proved unsuccessful except into the TL-4 cells monkeys. described above: 107 TL-4 cells were incubated for 2 h with 1 ml of p24-antigen positive supernatant from PB- RESULTS MCs of talapoin monkey 5, harvested 2 weeks after the Culture of talapoin monkey peripheral blood start of the culture. The infected TL-4 cells were ex- mononuclear cells panded and produced considerably more viral p24 antigen than the original PBMC cultures (Ͼ10-fold increase Previous attempts to isolate virus from the peripheral in OD value), until they were overgrown by CD8-express- blood mononuclear cells (PBMCs) of a number of sero- ing cells and stopped producing. Supernatant from these positive primate species have been unsuccessful. For cells was harvested and aliquoted; 1-ml aliquots were the most part, the experimental conditions were modeled used in the experimental infection of cynomolgus ma- on those optimized in the past for human cells, and our caques (see below). lack of success may have been due to the inability to properly propagate and stimulate the nonhuman PBMCs. Cloning of infected cells In this study, we evaluated a number of experimental conditions for viral isolation, including coculture with To enrich for virus producing cells, we have plated the human PBMCs or human cell lines, addition of irradiated antigen-producing PBMC cultures from talapoin 5 in 96- cells to provide growth factors, and titration of phytohe- well round-bottom plates at a density of 0.5 cell/well magglutinin (PHA) or interleukin-2 (IL-2) from different (cpw). No antigen-producing clones could be isolated sources. Using the optimum culture conditions (de- this way. Additional experiments included 10, 100, or scribed in the Materials and Methods), we have been 1000 cpw. Antigen-positive wells were found in the 100- able to propagate and expand PBMCs from talapoin and 1000-cpw cultures but not in the 10-cpw cultures. We monkeys for extended periods of time (months), although were unable to further expand the positive wells from biweekly removal of CD8ϩ cells remained necessary to either the 100- or 1000-cpw cultures. prevent overgrowth of the CD4ϩ cells. As an alternative, Electron microscopy we have attempted to transform talapoin monkey lymphocytes with human T cell leukemia virus type 1 The p24 cross-reactive antigen-producing PBMCs (HTLV-1) by cocultivating them with irradiated (6000 rad) were fixed and subjected to transmission electron mi- MT2 cells that produce (HTLV-1) (Markham et al., 1984). croscopy. The electron microscopic pictures (Fig. 1) iden- 118 OSTERHAUS ET AL.

human and nonhuman primate lentiviruses (Higgins et al., 1992). None of these monoclonal antibodies showed any reactivity toward SIVtal proteins.

Experimental infection Three experimental infections were performed. We initially inoculated one talapoin monkey (9) with 1 ml of culture supernatant from the original p24 antigen-producing PBMCs, taken 2 weeks after initiation of the culture. This animal seroconverted after 8 weeks. Virus could be isolated from this monkey only at 3 and 4 weeks postinfection (not shown). Western blot profiles from the sera of this monkey obtained at different times are shown in Fig. 3A. Antibodies cross-reactive with HIV-1 p24 were detected by 7 weeks p.i., and antibodies to multiple viral proteins by 52 weeks p.i. In the next exper- iment, we inoculated two cynomolgus macaques (monkeys 127 and 390) with 1 ml of culture supernatant of TL-4 cells that were infected in vitro with cell-free SIVtal. This supernatant is known to also contain HTLV-1 (see FIG. 1. Thin section electron micrograph of viral particles associated Genetic Analyses), and this could have resulted in coin- with PBMCs from talapoin monkey 5. The cells were harvested 2 weeks fection with both SIVtal and HTLV-1 in these animals. We after initiation of the culture, at which time viral antigen was detected did not test for HTLV-1 infection, and like in the SIVtal- in the supernatant. infected rhesus macaques, we found no deleterious con- sequences of the infection in these animals. Apparently, the presence of HTLV-1 did not accelerate or aggravate tified a lentivirus being produced by these cells, clearly SIVtal infection. Both of these monkeys seroconverted identifiable as a mature, enveloped virus particle of ap- (ELISA) at 8 weeks p.i. Western blot profiles of the sera of proximately 110 nM with a cone-shaped core and lateral these monkeys collected over time are displayed in Figs. bodies. No budding particles were observed. The virus is 3B and 3C; only cross-reactivity to HIV-1 p24 gag is tentatively named SIVtal (simian immunodeficiency virus observed in these animals during the follow-up period from talapoin monkeys). The number of cells surrounded by the observed particles was extremely small (Ͻ1:1000 cells), indicating the small number of cells actually infected and producing virus.

Protein profile Culture supernatant from p24-antigen-producing TL-4 cells infected with SIVtal was concentrated 1000-fold and purified on an Sephacryl S-500 column. A 4-␮l sample was loaded onto a 12.5% acrylamide gel (Phastsystem, Pharmacia) next to an HIV-1rf preparation prepared si- multaneously. The gel was blotted onto a nitrocellulose filter, reacted to pooled serum from three seropositive talapoin monkeys, and visualized using anti-human monoclonal antibody conjugated to alkaline phospha- tase (Sigma) and NBT/BCIP (Fig. 2). In the HIV-1 lane, the p39 partially cleaved gag precursor protein and the p24 matrix protein are detected. In the SIVtal lane, a p51 protein and a p26 matrix protein are detected. A number of bands of higher molecular weight are also detected in the SIVtal lane, presumably reflecting envelope gene products. We have also reacted identical SIVtal blot FIG. 2. Western blot of preparations of SIVtal (left lane) and HIV-1rf, strips to a panel of murine monoclonal antibodies char- reacted to sera from seropositive talapoin monkeys. See text for de- acterized previously that react to gag proteins of different tails. LENTIVIRUS FROM TALAPOIN MONKEYS 119

be necessary to develop cross-reactivity to other HIV-1 antigens.

CD4/CD8 ratios We have determined the reactivities of some murine monoclonal antibodies directed against human CD4 and CD8 antigens for reactivity with talapoin monkey PBMCs. Of the antibodies tested, we selected Q4084 (anti-CD4; Sattentau et al., 1989) and Leu2a (anti-CD8) because these two demonstrated the highest sensitivity and spec- ificity in reacting with talapoin monkey lymphocytes. Both Leu3A and OKT4 stained the same subset as that identified by Q4084 but with a lower fluorescence signal (data not shown). To determine the potential effect of SIVtal infection on T cell subset distributions in vivo in infected animals, we determined the percentages of CD4ϩ and CD8ϩ cells in the isolated PBMCs of talapoin monkeys that were either chronically infected (Ͼ2 years) or not infected with SIVtal. One talapoin monkey initially FIG. 3. HIV-1/2 Western blot strips (Cambridge Biotech) reacted to included in the uninfected group retrospectively demon- the sera from talapoin monkey 9 (A) and cynomolgus macaques 127 strated infection with SIVtal within 2 months after assaying and 390 (B and C, respectively). The size and origin of the reactive its CD4 and CD8 counts, indicating that infection may have bands are indicated at the left. Numbers at the top indicate the time point at which the serum samples were taken (in weeks postinfection). taken place around the time the assay was performed. This monkey is included in the “uninfected” group; exclusion of the data points obtained from this monkey did not influence (14 weeks p.i.). Virus could be reisolated from monkey the outcome of the statistical analyses. The results are shown in Table 1. Both the CD4 and CD8 percentages differ 390, but not from monkey 127, at 2 weeks p.i. (not shown). significantly between the monkeys in the “infected” and We then inoculated eight rhesus macaques according “uninfected” groups, which is also reflected in the CD4/CD8 to the scheme outlined below and in Fig. 4A. Two mon- ratios. Presumably, this is due to a decrease in CD4ϩ cells keys (negative controls) were inoculated with 1 million in the infected animals. uninfected PBMCs that had been harvested from talapoin monkey 9 before experimental infection and cul- Genetic analyses tured for 1 week. Two other monkeys received 1 million PBMCs from talapoin monkey 9 harvested 2 weeks post- Nucleic acids were extracted from the supernatant of experimental infection. After an additional 2 weeks, 10-ml SIVtal producing TL-4 cells and subjected to RT-PCR whole blood samples were taken from the two unin- using degenerate oligonucleotides specific for a highly fected and the two infected macaques and combined to conserved region of the polymerase gene (Wilson et al., give an infected pool and an uninfected pool. The two 1998). Three amplified fragments were obtained and se- pooled blood samples were transferred into two addi- quenced. The sequence of one fragment corresponded tional naive animals for each pool (see Fig. 4A). Blood to the published sequence for HTLV-1, which is not un- samples from all eight monkeys were analyzed for the expected because the TL-4 cells are derived from ta- presence of antibody and viral antigen using the isola- lapoin monkey PBMCs cocultured with HTLV-1-production procedures described above. Both monkeys that had ing MT-2 cells. The second fragment corresponded to a received cells from talapoin monkey 9 postinfection se- simian endogenous retroviral element (data not shown). roconverted after 6–8 weeks. Similarly, the two monkeys The third sequence (87-bp) clusters with lentiviral pol that had received blood samples from these two infected genes in phylogenetic analyses but is clearly different monkeys also seroconverted. Virus could be reisolated from all other lentiviral pol gene sequences published to from monkey 25280 at 10 weeks postinfection but not date and is believed to be representative of a new pri- from the other macaques (not shown). Western blot re- mate lentivirus provisionally named SIVtal, for SIV from activities of serum samples of all eight macaques taken talapoin monkeys. Alignment of the predicted amino acid at 0, 2, and 6 months postinfection are shown in Fig. 4B. sequences of the 87-bp SIVtal sequence and the homol- Antibody cross-reactivity to the HIV-1 major gag protein ogous sequences from selected primate lentiviruses (p24) is found in all four rhesus macaques that received suggests that SIVtal is different from all known primate the PBMCs from monkey 9 postinfection and not in the lentiviruses and potentially most related to the virus other monkeys. Prolonged follow-up of the animals may isolated from Sykes monkeys (not shown). 120 OSTERHAUS ET AL.

FIG. 4. (A) Schedule of experimental infection of rhesus macaques with SIVtal. (B) HIV-1 Western blot strips (Diagnostic Biotechnology) of sera from the monkeys in A. Three strips for each monkey reflect sera taken at 0, 2, and 6 months postinfection. Key (monkey/strip number): 25659/1–3; 25280/4–6; 26294/7–9; 26295/10–12; 25724/13–15; 25787/16–18; 25749/19–21; 25801/22–24; negative control/25, weak positive control/26, strong positive control/27.

Using a second set of primers, we obtained another ues at the bottom of the tree indicate that the exact 550-bp pol gene fragment. A comparison of this nucleo- phylogenetic relationships between SIVtal and other pri- tide sequence with the homologous sequences from mate lentiviruses could not be firmly established. The other primate lentiviruses is shown in Table 2. The SIVtal trees were obtained using neighbor joining methods; use sequence is roughly equidistant to the sequences from of maximum likelihood methods (for the nucleotide se- all other primate lentiviruses, with nucleotide identities quences) yielded a tree with the same topology. within this fragment varying between 57% and 62% and amino acid identities between the predicted translation DISCUSSION products varying between 49% and 57%. Phylogenetic analysis of this sequence is shown in Figs. 5A (nucleo- We have identified in captive talapoin monkeys a new tides) and 5B (amino acids). Based on those trees, SIVtal member of the growing family of nonhuman primate appears to be a separate subspecies of primate lentivi- lentiviruses. Genetic analyses of two fragments of the ruses that branches off the root of the tree and forms a polymerase gene demonstrate that this virus is different branch that possibly includes SIVsyk. Low bootstrap val- from all primate lentiviruses identified to date. Six genet- LENTIVIRUS FROM TALAPOIN MONKEYS 121

TABLE 1 Percentages of CD4ϩ and CD8ϩ Cells in Isolated PBMCs of Talapoin Monkeys, Related to Infection Status

CD4 CD8 CD4/CD8 Infection status Talapoin Infected Uninfected Infected Uninfected Infected Uninfected

1 21 89 0.2 6 34 65 0.5 2 54 36 1.5 3 63 N.D. N.D. 4 52 35 1.5 7a 57 30 1.9 jm 43 42 1.0 P ϩ vs ϪϽ.005 Ͻ.005 Ͻ.05

Note. Values reflect percentages P values are determined using the Student’s t test. a Talapoin monkey 7 seroconverted within 2 months before the analysis and was included in the “uninfected” group. ically related groups of primate lentiviruses have been genetic group or whether they define two separate previously identified: the HIV-1/SIVcpz group (including groups (Fig. 5). More sequence data are needed to fully SIVcpz, HIV-1 M, and O clades) with a subgroup contain- classify SIVtal with respect to the other primate lentivi- ing the drill and red-capped mangabey viruses (SIVdrl, ruses. SIVrcm), the HIV-2-SIVsm group, the SIVagm group, the Previous attempts at passaging SIVtal in vitro or in vivo SIVmnd group containing the mandrill and L’Hoest mon- have been unsuccessful (monkey 22). We have now key viruses, and the SIVsyk group. Of the last group, only succeeded in obtaining one isolate of this virus from one one member is known. In addition, a virus that appears to infected monkey. The virus is infectious in vivo in talapoin be a recombinant between ancient members of the HIV-2 monkeys and in rhesus and cynomolgus macaques, as and SIVagm groups has been described in sabaues demonstrated by seroreactivity and virus reisolation. monkeys (Jin et al., 1994) Preliminary phylogenetic anal- Propagation of SIVtal in tissue culture is difficult: infected yses indicate that the newly identified virus, designated PBMCs can be propagated and produce small amounts SIVtal, possibly defines a sixth group, distantly related to of virus, but the addition of uninfected talapoin monkey all other groups. The sequence we have determined is PBMCs apparently does not result in infection of more relatively small, and care must be exercised in interpret- cells or higher virus production. We have not been suc- ing the genetic differences with those of other lentivi- cessful in transferring SIVtal to either primary human ruses. Table 2 shows a comparison between the homol- cells or cultured continuous human T cell lines. This may ogous sequences from different primate (human and reflect a total inability to replicate in human cells or nonhuman) lentiviruses. Based on this comparison, the alternatively may reflect the poor transmissibility of this closest relative of SIVtal appears to be SIVsyk; however, virus in vitro because infection of talapoin monkey it is not clear whether these viruses belong to the same PBMCs by SIVtal in vitro is also highly inefficient. If the

TABLE 2 Percent Nucleotide and (Amino Acid) Identities between the 550-bp pol Gene Fragments of Selected Primate Lentiviruses

Virus SIVsyk SIVsm HIV-2rod SIVsab SIVtan SIVagm667 SIVagm3 SIVhst SIVmnd SIVdrl HIV-1ant70 SIVcpz HIV-1jrcsf

SIVtal 60 (56) 58 (52) 58 (50) 62 (57) 60 (55) 61 (56) 62 (53) 59 (50) 60 (50) 57 (53) 58 (51) 59 (49) 58 (50) SIVsyk 59 (56) 58 (54) 60 (54) 60 (58) 58 (53) 58 (51) 58 (53) 59 (53) 56 (53) 57 (53) 60 (54) 57 (53) SIVsm 78 (86) 65 (64) 56 (56) 59 (58) 56 (54) 61 (57) 59 (57) 63 (60) 62 (59) 63 (60) 65 (59) HIV-2rod 64 (64) 58 (55) 62 (57) 57 (55) 60 (55) 61 (55) 63 (59) 64 (59) 63 (59) 63 (56) SIVsab 63 (60) 60 (61) 63 (61) 62 (54) 65 (58) 66 (66) 67 (61) 66 (59) 64 (58) SIVtan 66 (68) 70 (70) 67 (64) 66 (65) 62 (64) 63 (61) 64 (63) 62 (62) SIVagm667 67 (72) 59 (57) 59 (57) 61 (63) 62 (60) 62 (59) 61 (59) SIVagm3 63 (58) 67 (63) 62 (57) 62 (57) 64 (60) 62 (56) SIVhst 70 (77) 59 (56) 62 (59) 63 (58) 60 (56) SIVmnd 63 (59) 66 (62) 66 (61) 63 (56) SIVdrl 71 (72) 68 (70) 67 (67) HIV-1ant70 78 (85) 75 (79) SIVcpz 81 (85) 122 OSTERHAUS ET AL.

difficult due to rapid overgrowth by CD8-bearing cells, which did not produce SIVtal. The inability to enrich our in vitro cell cultures for virus-producing cells appears to coincide with enhanced cell death in these cultures, although cell viability was not measured quantitatively. No cytopathic effects attrib- utable to SIVtal were observed in infected cultures, which is not surprising in light of the small number of cells infected. Interestingly, talapoin monkeys that had been infected with SIVtal for more than 2 years showed reduced percentages of circulating CD4ϩ cells and con- comitantly increased percentages of CD8ϩ cells. So far, all known primate lentiviruses have been nonpathogenic in their natural hosts. Despite the apparent gradual loss of CD4ϩ cells, we have not seen any disease associated with SIVtal infection of talapoin monkeys. The situation with SIVtal is reminiscent of feline immunodeficiency virus infection in cats, where CD4 depletion is observed both in vivo and in vitro, but immunodeficiency-related disease is apparent only later in life. Potentially, SIVtal may cause disease in older, chronically infected talapoin monkeys. No sign of CD4 depletion was seen in the four SIVtal-infected rhesus macaques in the 6-month follow-up period after experimental infection. Although it is not known whether SIVtal infection of talapoin monkeys occurs in the wild, the high incidence of HIV-1 cross-reactive antibodies in captive talapoin monkeys maintained in different parts of the world suggests that these animals are naturally infected with a lentivirus, herein denoted SIVtal. Monitoring the colony in the Netherlands provided evidence for active transmission of virus because more animals became positive over time. This seroconversion of previously seronegative monkeys that were housed together provides evidence for horizontal transmission of SIVtal. Further eval- uation of SIVtal infection in various nonhuman primate FIG. 5. Phylogenetic trees of the 550-bp pol gene fragments (A) and species, together with analyses of the SIVtal genome predicted amino acid translations (B) of selected primate lentiviruses. Numbers at branchpoints represent bootstrap values. Only bootstrap may provide further insight into the mechanisms of len- values of more than 50 are shown. GenBank accession numbers: tivirus pathogenicity. SIVsab, U04018; HIV-1vau/ham, Y14505; HIV-192RW009, AF009408; SIVdrl, AJ011017; SIVsab, U04018; SIVtan, U58991; SIVhst, AF075269. MATERIALS AND METHODS All talapoin monkeys in this study were part of a group former is true, then this virus may not pose an immediate of nine animals at the Amsterdam Zoo Artis. Two of the threat to humans, despite frequent interactions between animals were born in Artis, and the other seven came to humans and talapoin monkeys. Cocultivation of talapoin Artis via other European zoos. Of these seven, two were monkey PBMCs with HTLV-1-transformed MT-2 cells originally wild-caught. Talapoin monkey 5 was a female, yielded talapoin monkey T cells with less stringent approximately 4 years of age at the time of first virus growth requirements. We have not attempted to deter- isolation, and talapoin monkey 9 was a female, approx- mine whether these cells are actually HTLV-1 trans- imately 11 years of age at time of experimental infection. formed, but the fact that these cells still produce HTLV-1 Culture of talapoin monkey PBMCs after 2 months of cocultivation suggests productive infection with HTLV-1. These cells (TL-4 cells) appear to be PBMCs were isolated from the blood of talapoin mon- infectable with SIVtal and, on infection, produce relatively keys by density centrifugation onto a dextran 500/sodium large amounts of virus. However, long-term propagation metrizoate (4:10.5%) cushion. The cells were washed and of virus-producing cultures of TL-4 cells proved to be resuspended in culture medium (RPMI/1640 containing LENTIVIRUS FROM TALAPOIN MONKEYS 123

10% FCS (Hyclone) and supplemented with penicillin, regions in the polymerase gene that are conserved be- streptomycin, and L-glutamine. The cells were stimulated tween HIV-1, HIV-2, and SIV (5Ј-TATTAGAYACAGGGGCW- for 3 days with 100 ␮g/ml PHA (Wellcome), washed, and GAYGA and 5Ј-AGGGWAYGGAAAARTAKGCATC). The then cultured in the presence of 50 ␮g/ml PHA and 10 two fragments are 57 and 550 nucleotides in length, U/ml IL-2 (Biotest). Irradiated (3000 rad) CEMx174 cells respectively. Phylogenetic analysis was performed on were added to these cultures as feeder cells at a 1:10 the 550-bp fragment using neighbor joining algorithms ratio. For HTLV-1 transformation, 106 PHA-stimulated ta- on the nucleotide alignments. Alignments were per- lapoin monkey PBMCs were cocultured with 105 irradi- formed using CLUSTAL-W (Higgins and Sharp, 1988; ated (6000 rad) MT2 cells (MRC AIDS Reagent Reposi- Thompson et al., 1994), and phylogenetic analyses were tory) in the presence of 10 U/ml IL-2. performed using the programs SEQBOOT, DNADIST (or PROTDIST), NEIGHBOR, and CONSENSE from the Virus and antibody detection PHYLIP suite of programs for phylogenetic analysis (J. Viral antigen was detected in tissue culture superna- Felsenstein, University of Washington). Both sequences tant using a polyclonal HIV-1 p24-antigen capture ELISA were submitted to GenBank and can be accessed using (Organon Teknika). Antiviral antibodies in the serum of accession numbers AF119257 and AF119258. infected animals were detected using a commercial HIV-1 antibody detection ELISA (Coulter). Western blots REFERENCES were performed on HIV-1 Western blot strips (Cambridge BioTech and Diagnostic Biotechnology) at 1:100 serum Allan, J. 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