Isolation and Characterization of Simian Immunodeficiency Virus

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JOURNAL OF VIROLOGY, Nov. 1988, p. 4044-4050 Vol. 62, No. 11 0022-538X/88/114044-07$02.00/0 Copyright C 1988, American Society for Microbiology Isolation and Characterization of Simian Immunodeficiency Virus from Mandrills in Africa and Its Relationship to Other Human and Simian Immunodeficiency Viruses HAJIME TSUJIMOTO,'* ROBERT W. COOPER,2 TOSHIAKI KODAMA,1 MASASHI FUKASAWA,1 TOMOYUKI MIURA,1 YOSHIHIRO OHTA,l KOH-ICHI ISHIKAWA,1 MASUYO NAKAI,3 ERIC FROST,2 GEORGE E. ROELANTS,' JEAN ROFFI,2 AND MASANORI HAYAMI' Department ofAnimal Pathology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108,1 and Osaka Medical College, Takatsuki, Osaka 569,3 Japan, and Centre International de Recherches Medicales de Franceville, B.P. 769, Franceville, Gabon2 Received 15 April 1988/Accepted 13 July 1988

Two isolates of simian retrovirus related to the human immunodeficiency virus (HIV) were obtained from apparently healthy mandrills, Papio (Mandrillus) sphinx, in western equatorial Africa. This virus, designated SIVMND (simian immunodeficiency virus from mandrills), appeared morphologically similar to HIV by electron microscopy, showed Mg2 -dependent reverse transcriptase activity, and induced cytopathic effect in human CD4-positive cells. Western blotting (immunoblotting) analyses revealed that the gag and pol products of SIVMND showed cross-reactivity with those of known HIVs and SIVs. Molecular clones covering full-length viral DNA were obtained from closed circular extrachromosomal DNA of SIVMND-infected cells. By clone-on-clone hybridization with known retroviruses of the HIV and SIV groups, SIVMND showed similar cross-hybridization with HIV-1, HIV-2, SIVAGM (African green monkey-derived SIV), and SIVMAC (rhesus macaque-derived SIV) in the gag and pol regions only at low stringency but not at high stringency, a result indicating that SIVMND is a new member of the HIV-SIV group. The existence of distinct SIVs in different monkey species suggest that recent interspecies transfer of HIV-SIV is unlikely in nature.

The epidemic of acquired immunodeficiency syndrome obtain isolates from many more species of naturally infected (AIDS) began in 1981 in the United States and apparently as primates atid to study their genetic relationship. Herein we early as the mid-1970s in Central Africa and has now spread report the isolation of a new SIV from wild-caught mandrills, worldwide (6, 31). Human immunodeficiency virus type 1 Papio (Mandrillus) sphinx, in Gabon, the cloning of the (HIV-1) belonging to the lentivirus family (13), which was entire viral genome, and its comparison with other viruses of formerly called lymphadenopathy-associated virus (2), hu- the HIV and SIV groups. man T-lymphotropic virus type III (30), or AIDS-associated retrovirus (24), is kniown to be an etiological agent of AIDS MATERIALS AND METHODS in humans. A simian retrovirus antigenically related to HIV was first Animals. Of 16 wild-caught mandrills in a semi-free-range isolated from a rhesus macaque, Macaca mulatta, with an breeding colony maintained by the Primatology Center of the AIDS-like syndrome at a U.S. primate center (7, 22). The Centre International de Recherches Medicales de France- virus was initially designated as rhesus macaque-derived ville in Gabon, two adult animals (a breeding male and simian T-lymphotropic virus type III but is now called female) were found to be weakly seropositive for SIV from rhesus macaque-derived simian immunodeficiency virus, African green monkey, SIVAGM, by immunofluorescence SIVMAC. Subsequently, a large proportion of African green and Western blotting (immunoblotting) analyses (28). Be- monkeys (Cercopithecus aethiops) (16, 20, 21, 28) and sooty cause these adults had been seropositive since their acqui- mangabey monkeys (Cercocebus atys) (12, 26, 27) were sition by the center as juveniles in 1979 and 1983, respec- shown to be naturally infected with HIV-related retroviruses tively, it is virtually certain that they had become infected in but were apparently free of AIDS-like disease. A second the wild. All animals in this colony, including these two human AIDS-related retrovirus, HIV-2, isolated from pa- SIV-seropositive adults, were healthy and with no symp- tients with AIDS in West Africa, was shown to be antigen- toms of AIDS-like disease. ically closer to SIVMAC than to HIV-1 (5). Nucleotide Virus isolation. Peripheral-blood mononuclear cells from sequence analyses indicate that HIV-2 is closely related to the two seropositive mandrills (2 x 106 cells per ml) were SIVMAC, with about 75% overall nucleotide sequence ho- stimulated with 25 4L of concanavalin A per ml for 24 h and mology, but only distantly related to HIV-1, with about 40% cultured in RPMI 1640 growth medium supplemented with overall homology (4, 10, 14). 20% heat-inactivated fetal calf serum, antibiotics, and 10% On the basis of these observations, some workers sug- crude interleukin-2. After cultivation for 3 to 7 days, these gested that monkeys might be a reservoir for an etiological mandrill cells were cocultivated with Molt-4 clone 8 cells agent of AIDS in humans (20, 21, 27). To understand the (23), which are highly susceptible to HIV-1 infection and its origin, natural history, and evolutionary relationships of cytopathogenicity. After cocultivation, the medium without viruses of the HIV and SIV groups, it will be necessary to interleukin-2 was replaced every 3 days, and the cultures were examined for the expression of cytopathic effects (CPE). The expression of viral proteins in cultured cells was * Corresponding author. detected by immunofluorescence assay as described previ- 4044 VOL. 62, 1988 SIMIAN IMMUNODEFICIENCY VIRUS FROM MANDRILLS 4045

ously (28). The release of virus particles was examined by chloroform-isoamyl alcohol. The XbaI-digested DNA frac- reverse transcriptase (RT) assay (28) and electron micros- tion containing linearized SIVMND DNA was ligated with copy. When the production of SIV in the Molt-4 clone 8 cells XbaI-digested lambda-ong-C vector arms (Stratagene, San was confirmed, the supernatant was inoculated into un- Diego, Calif.) and packaged in vitro with Gigapack Gold cloned Molt-4 cells to obtain a persistently virus-infected cell (Stratagene), and the recombinant phages were plated and line showing no CPE. screened in situ with a 32P-labeled full-length SIVAGM probe Western blotting. For both mandrill isolates, virions were after transfer to nylon membrane filters. Several signal- concentrated and purified from the culture supernatant of the positive plaques were purified and expanded, and the insert persistently virus-infected Molt-4 cells by sucrose gradient DNA in phage vector was cut out by XbaI digestion and ultracentrifugation as described previously (28). HIV-1 and recloned into a pUC119 plasmid vector (33). SIVAGM virions were also prepared from H9 cells infected Southern blot hybridization. Undigested and restriction with human T-lymphotropic virus type "IB (30) and Molt-4 endonuclease-digested DNA samples were subjected to elec- cells infected with SIVAGM[TYO-1] (previously called SIV trophoresis in 0.8% agarose gel and transferred to nylon [AGM-1]) (28), respectively, by the same procedures. These membrane filters. The filters were then irradiated with UV virion suspensions were treated with 2% sodium dodecyl ight to cross-link them to the DNAs. Hybridization at low sulfate and 5% 2-mercaptoethanol, boiled briefly, and sub- stringency was carried out at 37°C in a mixture of 6x SSC jected to electrophoresis on 12% polyacrylamide slab gels. (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 25% After electrophoresis, the size-fractionated proteins were formamide, 5x Denhardt solution, 0.1% sodium dodecyl transferred to Durapore filters (Millipore Corp., Bedford, sulfate, 10 mM EDTA, 20 ,ug of denatured salmon sperm Mass.) by electroblotting. The filters were incubated with DNA per ml, and a 32P-labeled probe (2 x 106 to 5 x 106 5% fetal calf serum to block nonspecific reactions and then cpm/ml). After hybridization, the filters were washed three cut into strips. The strips were incubated overnight at 4°C in times at 50°C with a solution containing 2x SSC, 0.1% a 1:100 dilution of human or monkey serum. Then, the strips sodium dodecyl sulfate, and 1 mM EDTA. This low-strin- were washed and incubated with biotin-labeled sheep anti- gency condition corresponds to Tm - 40°C, a value calcu- human immunoglobulins at 37°C for 1 h and then with lated by using the standard formula and taking the GC biotinylated peroxidase-streptoavidin complex (Amersham, content of HIV-1 as 42% (34). A hybridization solution Tokyo, Japan) at room temperature for 30 min. The strips containing 30% formamide at 37°C and a washing solution were then washed, and peroxidase activity was detected by containing 0.5x SSC at 50°C were used for a medium- the addition of a substrate solution containing 0.5 mg of stringency condition (Tm - 30°C), and 40% formamide diaminobenzidine per ml supplemented with 0.01% hydro- hybridization solution at 37°C and a washing solution con- gen peroxide. taining 0.2x SSC at 55°C were used for a high-stringency DNA clones of reference virus strains. A 32P-labeled condition (Tm - 23°C). These filters were then exposed to pSAH12 probe was used to detect the genome of simian X-ray films for 12 to 36 h at -70°C. immunodeficiency virus from mandrills (SIVMND) for molecular cloning. This probe is a full-length clone of SIVAGM RESULTS isolated in our laboratory (11). Molecular clones of HIV-1, HIV-2, SIVMAC, and SIVAGM were used for comparison Virus isolation. Peripheral-blood mononuclear cells from with SIVMND. A nearly full-length DNA clone of HIV-1 the two seropositive mandrills were cultured for 3 to 7 days containing a 9-kilobase (kb) SacI fragment (pBT-1) (1) was and then cocultivated with Molt-4 clone 8 cells. After kindly provided by Luc Montagnier. The HIV-2 clone used cocultivation for 1 to 2 weeks, characteristic giant-cell was pGH-1, which covers the entire genome of HIV-2[GH-1] formation like that in HIV-infected Molt-4 clone 8 cells was and was isolated from a patient with AIDS in Ghana (K. observed in both cocultures from the mandrills. On immu- Ishikawa, H. Tsujimoto, M. Nakai, J. A. A. Mingle, M. nofluorescence assay, these giant cells and some normal- Osei-Kwasi, S. E. Aggrey, V. B. A. Nettey, S. N. Afoakwa, sized cells gave strong positive reactions with sera from M. Fukasawa, T. Kodama, M. Kawamura, and M. Hayami, these two seropositive mandrills but only weakly positive manuscript submitted for publication); its approximate nu- reactions with anti-HIV-1-positive human sera and anti- cleotide sequence homology with the Institute Pasteur iso- SIVAGM-positive African green monkey sera. late, HIV-2RoD (14), is 90% in the gag and pol regions and The culture supernatants of these Molt-4 clone 8 cells 80% in the env region. The SIVMAC (simian T-lymphotropic showing marked CPE contained high Mg2'-dependent RT virus type III) probes used were pK2-lOBamA (gag-pot) (18) activity. The RT activity reached a maximum of more than 1 and pK2-OLTR (3' open reading frame-long terminal repeat x 106 cpm/ml, and the RT activity with Mg2+ was more than [LTR]), which were kindly provided by James I. Mullins. 10 times that with Mn2+. Electron microscopic examination Molecular cloning. Extrachromosomal DNA was prepared of the cells showing CPE revealed mature virus particles of essentially by the Hirt method (19) from a 24-h culture of 100- to 120-nm diameter in the extracellular space (Fig. 1). Molt-4 clone 8 cells inoculated with 104 to 105 50% tissue The mature particles consisted of a condensed core that culture infective dose per ml of SIVMND[GB-1] and showing appeared tubular-prismatic or centrosymmetric, depending prominent CPE such as syncytium formation and ballooning. on the plane of section, and an envelope with relatively The DNA sample was treated with proteinase K (50 ,ug/ml) prominent knobs. Particles budding from the cell membrane at 37°C for 2 h, extracted with phenol and chloroform- in which the electron-dense shell was closely apposed to the isoamyl alcohol (24:1), precipitated with ethanol, and then envelope were also seen. These two SIV isolates were treated with RNase A (50 p.g/ml). Closed circular DNA of designated SIVMND[GB-1 and SIVMND[GB-2]. SIVMND, which was shown to have a single XbaI site in the As CPE in the Molt-4 clone 8 cells became prominent, cell circular form, was linearized by digestion with XbaI. The proliferation became markedly impaired. Therefore, the linearized viral DNA was fractionated by 0.8% agarose gel supernatant of the infected cells was inoculated into un- electrophoresis, and the DNA fraction of 8 to 10 kb was cloned Molt-4 cells to obtain persistently infected cell lines. electroeluted from the gel and extracted with phenol and At 1 or 2 weeks after inoculation, the Molt-4 cells showed 4046 TSUJIMOTO ET AL. J. VIROL.

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CPE which was not so marked as that for clone 8 cells. After ucts of SIVMND showed distinct cross-reactivity with those 4 to 5 weeks, the CPE disappeared, and normal cell prolif- of HIV-1 and SIVAGM but that the env gene products of eration resumed, but almost all the cells from the two Molt-4 SIVMND partially cross-reacted only with those of SIVAGM. cell lines expressed viral antigens that were detected with Furthermore, the molecular weights of a gag protein (p26) seropositive mandrill sera by immunofluorescence. Further- and transmembrane protein (gp3O) in SIVMND were slightly more, high RT activity and abundant virus particles were different from their corresponding proteins in HIV-1 (9) and detected in these cell cultures. SIVAGM (28). Analysis of virion proteins. The purified virions of two Molecular cloning. Immunologic cross-reactivity between SIVMND isolates collected from the culture supernatant of SIVMND and SIVAGM indicated the possible value of cloned persistently infected Molt-4 cells were analyzed by Western SIVAGM DNA as a probe for detecting the SIVMND genome. blotting for identification of the SIVMND proteins and their Hirt supernatant DNA prepared from Molt-4 clone 8 cells cross-reactivity with HIV-1 and SIVAGM (Fig. 2). When the showing marked CPE after infection with SIVMND was virion proteins of SIVMND were incubated with the serum of subjected to Southern blot hybridization with an SIVAGM a patient with AIDS containing anti-HIV-1 antibody, four full-length probe, pSAH12. major bands at positions of approximately 66, 55, 31, and 26 With these DNAs, faint specific hybridizing bands were kilodaltons, corresponding to the pol and gag gene products detected only at low stringency (Tm - 40'C) after a long of HIV-1 (8, 25), were specifically identified. Anti-SIVAGM- exposure (Fig. 3A) and not at medium (Tm - 30°C) or high positive African green monkey serum reacted with the stringency. The undigested Hirt supernatant gave a band of protein of a smeared band at approximately 30 kilodaltons, approximately 9.4 kb and two very faint bands of about 5 kb. corresponding to the gp32 transmembrane protein of Probably, the former band corresponded to linear uninte- SIVAGM (28), and with the gag and pol gene products of grated viral DNA, whereas the latter bands originated from SIVMND. The serum of the mandrill from which the closed circular forms with one and two LTRs (29). After SIVMND[GB-1 was isolated also recognized a 120 kilodal- XbaI digestion, the two bands of closed circular DNA were ton-protein of SIVMND, which should correspond to the shifted to a position of about 9.4 kb, and the linear viral DNA surface protein of the envelope. The mandrill serum reacted was cut into two fragments of 5.6 and 3.8 kb, a result mainly with env gene products and scarcely reacted with the indicating that XbaI has a single cutting site in the SIVMND gag and pol gene products of even SIVMND. These Western genome. The XbaI-digested Hirt supernatant DNA of blotting analyses indicated that the gag and pol gene prod- SIVMND-infected cells was ligated to XbaI-digested lambda- VOL. 62, 1988 SIMIAN IMMUNODEFICIENCY VIRUS FROM MANDRILLS 4047

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FIG. 2. Identification of SIVMND proteins and their cross-reactivity with those of HIV-1 and SIVAGM by Western blot analysis. Purified virions of SIVMND[GB-1] (lanes 1), SIVMND[GB-2] (lanes 2), SIVAGM (28) (lanes 3), and HIV-1 (30) (lanes 4) were examined by Western blot analysis with sera from a patient with AIDS and seropositive for HIV-1 (A), an African green monkey seropositive for SIVAGM (B), and the mandrill from which SIVMND[GB-1] was isolated (C). ong-C vector arms and packaged. Of about 5 x 104 plaques, (A) (B) 10 clones showed a positive signal on in situ screening with the SIVAGM probe under conditions of low stringency. Of kb 1 2 1 2 3 4 these clones, eight had an insert of 8.8 kb and two had an kb insert of 9.5 kb. Restriction endonuclease mapping indicated 23- that the clone with the longer insert (pSMH103) had two 23 - additional adjoining PstI and XhoI sites. Thus the long and short inserts may correspond to the full-length SIVMND genome with two and one LTRs, respectively. 9.4 - ",: 9.4 - la Detection of viral genome in infected cells. Hirt supernatant DNA from Molt-4 clone 8 cells infected with SIVMND[GB- 6.6- 6.6-X 1], which was used as a DNA source for molecular cloning, was examined by Southern blot hybridization with the 32P-labeled cloned SIVMND insert, pSMH103 (Fig. 3B). The 4.4- 4.4- _0 undigested DNA gave strong bands of approximately 9.4 kb and two faint bands of about 5 kb, and the digests with several restriction endonucleases also gave distinct bands at high stringency (Tm - 23°C), which coincided with the faint bands detected with the SIVAGM probe at low stringency. 2.3- 2.3 - The SIVMND genome in the chromosomal DNA of the two 2.0- 2.0- el P Molt-4 cell lines persistently infected with SIVMND[GB-1l and SIVMND[GB-2] was then examined. For both of these cell lines, bands that hybridized distinctly and corresponded in size to internal fragments of the cloned SIVMND genome were detected under conditions of high stringency. The S. DNA fragmentation patterns of these two cell lines with BamHI, PstI, Hindlll and XbaI were identical. These SIVMND-infected cell DNAs did not give any detectable FIG. 3. Detection of unintegrated viral DNA in SIVMND-infected bands when they were probed with cloned HIV-1, HIV-2, cell line. (A) Hirt supernatant DNA prepared from approximately 1 SIVMAC, or SIVAGM under the same conditions. x 107 Molt-4 clone 8 cells infected with SIVMND[GB-1] (lane 1) and Homology between SIVMND and other viruses of the HIV its digest with XbaI (lane 2) were subjected to Southern blotting and SIV groups. The approximate sequence homologies analysis with a 32P-labeled full-length clone of SIVAGM, pSAH12, under conditions of low stringency (Tm - 40°C). (B) The same Hirt between SIVMND and known viruses of the HIV and SIV supernatant DNA preparation was blot analyzed by using a 32p_ groups were then examined. First, full-length or nearly labeled SIVMND full-length clone, pSMH103, under conditions of full-length DNAs of HIV-1 (pBT-1), HIV-2 (pGH-1), high stringency (Tm - 23°C). Results for undigested Hirt supernatant SIVMAC (pK2-lOBamA), and SIVAGM (pSAH12) excised (lane 1) and its digests with XbaI (lane 2), BamHI (lane 3), and from plasmid clones were blot hybridized to a 32P-labeled EcoRI (lane 4) are shown. 4048 TSUJIMOTO ET AL. J. VIROL.

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--pot eenv qag pol- FIG. 4. Examination of SIVMND homology with known HIVs and SIVs. (A) Cross-hybridization experiment under various degrees of stringency. The insert DNAs (0.5 p.g per lane) of the virus clones stained with ethidium bromide are shown on the top of the panel. Lanes: 1, pSMH103 from SIVMND; 2, pBT-1 from HIV-1; 3, pGH-1 from HIV-2; 4, pK2-l0BamA from SIVMAC; 5, pSAH12 from SIVAGM. These clones cover almost the entire genome of each virus, except for the SIVMAC clone which does not contain LTR. These cloned DNAs (10 ng per lane for SIVMND and 100 ng per lane for other clones) were subjected to Southern blot hybridization with the 32P-labeled pSMH103 probe, under conditions of low stringency (Tm - 40°C) (a), medium stringency (Tm, - 30°C) (b), and high stringency (Tm - 23°C) (c). (B) Cross-hybridization between SIVMND and HIV-1 in their subgenomic regions. An ethidium bromide-stained pSMH103 (0.5 p.g per lane) digested with BamHI-Xhol (lanes 1), PstI (lanes 2), or Kpn-Sacl (lanes 3) after digestion with Xbal is presented in subpanel a. The digests were hybridized under conditions of low stringency with one of four subgenomic probes of pBT-1 digested with HincII: b, gag-pol; c, pol-sor; d, 5' env; e, 3' env-LTR. (C) Restriction map of pSMH103 (SMH 103). Regions showing cross-hybridization between SIVMND and HIV-1 are shaded. Abbreviations: B, BamHl; K, KpnI; P, PstI; S, Sacl; Xb, XbaI; Xh, XhoI.

SIVMND full-length probe (pSMH103) under various degrees by HincIl digestion of pBT-1. Distinct bands were detected of stringency (Fig. 4A). Under low stringency (Tm - 40°C), when the SIVMND DNAs were probed with labeled subge- distinct cross-hybridization was observed between SIVMND nomic probes of pBT-1 that corresponded to the gag and pol and all of the viruses HIV-1, HIV-2, SIVMAC, and SIVAGM. regions. In the case of SIVMND DNA digested with BamHI However, under medium- (Tm - 30°C) and high-stringency and XhoI, a 2.5-kb fragment hybridized with the HIV-1 (Tm - 23°C) conditions, cross-hybridization was very faint gag-pol probe, and 3.7- and 1.0-kb fragments hybridized or insignificant, a result indicating that the approximate with the pol-sor probe (Fig. 4B). This cross-hybridization sequence homology between SIVMND and the other four data indicated that the gag and pol regions in the SIVMND HIV or SIV clones was only 50 to 70% even in the regions genome had approximately 50 to 70% homology with those showing cross-hybridization. These results provided no ev- of HIV-1, whereas the homologies in other regions such as idence that SIVMND cross-hybridized especially strongly env and LTR were lower. From this experiment, putative with any one of these HIVs or SIVs. On the basis of these gag and pol genes could be localized in the cloned SIVMND data, the SIVMND isolated and molecularly cloned in this genome by their homology with the corresponding subgeno- study can be regarded as a new virus belonging to the mic fragments of HIV-1. Furthermore, clone-on-clone -hy- HIV-SIV group. bridization experiments of SIVMND DNA with HIV-2, To localize the regions in the SIVMND genome that cross- SIVMAC, and SIVAGM probes indicated that SIVMND also hybridized with HIV-1, DNA fragments of SIVMND has approximately 50 to 70% sequence homology with all of (pSMH103) digested with appropriate restriction enzymes these viruses in the gag and pol regions (data not shown), were blot hybridized to 32P-labeled subgenomic DNAs of just as demonstrated with HIV-1. Thus SIVMND was shown HIV-1 under conditions of low stringency (Fig. 4B). Four to be approximately equidistantly related to HIV-1, HIV-2, kinds of subgenomic DNA probes of HIV-1 were prepared SIVMAC, and SIVAGM. VOL. 62, 1988 SIMIAN IMMUNODEFICIENCY VIRUS FROM MANDRILLS 4049

DISCUSSION relationships of the viruses of the HIV and SIV groups, further efforts will be required to isolate and characterize The present study provided convincing evidence that new SIVs from as many sources as possible. mandrills are naturally infected with a retrovirus that is genetically related to HIVs and SIVs but clearly distinct ACKNOWLEDGMENTS from all other viruses of the HIV and SIV groups that were This work was partly supported by a Grant-in-Aid for Cancer examined. Research from the Ministry of Education, Science and Culture of Because of antigenic similarities or common African gen- Japan. Centre International de Recherches Medicales de Franceville esis, it has been suggested that HIV may have originated is supported by the State of Gabon and Elf Gabon. from SIV of African monkeys (20, 21, 27). However, this The invaluable assistance of P. Bussi, A. T. C. Feistner, F. study and our previous studies of SIV (11, 28) do not support Moysan, and N. Orbell in handling the mandrills is greatly appreci- this notion. We have shown by using genomic comparisons ated. that SIVs isolated from mandrills, African green monkeys, LITERATURE CITED and rhesus macaques are all quite distinct. Furthermore, these SIVs are distantly related to both HIV-1 and HIV-2, 1. Alizon, M., P. Sonigo, F. Barre-Sinoussi, J. C. Chermann, P. except for SIVMAC, which showed close relatedness to Tiollais, L. Montagnier, and S. Wain-Hobson. 1984. Molecular cloning of lymphadenopathy-associated virus. Nature (London) HIV-2. Although the interspecies transfer of HIVs and SIVs 312:757-760. in artificial situations cannot be denied, infections by these 2. Barr&Sinoussi, F., J. C. Chermann, F. Rey, M. T. Nugeyre, S. viruses seem to be limited to their natural hosts. Chamaret, J. Gruest, C. Dauguet, C. Axier-Blin, F. Brun- In this study, the SIVMND strains were isolated from Vezinet, C. Rouzioux, W. Rozenbauni, and L. Montagnier. 1983. mandrills inhabiting western parts of equatorial Africa such Isolation of a T-lymphotropic retrovirus from a patient at risk as Gabon, the Cameroons, and the Congo (35). On the other for acquired immune deficiency syndrome (AIDS). Science 220: hand, SIVAGM isolates were obtained from African green 868-871. monkeys originally captured in parts of eastern Africa such 3. Benveniste, R. E., L. 0. Arthur, C. Tsai, R. Sdwder, T. D. L. E. and S. 1986. Isolation of as Ethiopia and Kenya (28). Seropositive African green Copeland, Henderson, Oroszlan. a lentivirus from a macaque with lymphoma: comparison with monkeys have been reported before from this region (20, 21). HTLV-III/LAV and other lentiviruses. J. Virol. 60:483-490. These monkeys appear to have been naturally infected with 4. Chakrabarti, L., M. Guyader, M. Alizon, M. D. Daniel, R. C. SIVs from olden times, before the AIDS epidemic in humans Desrosiers, P. Tiollais, and P. Sonigo. 1987. Sequence of simian (17). 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