Virology 260, 116–124 (1999) Article ID viro.1999.9794, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE 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,i,3 and Marnix L. Bosch*,i,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 iRegional 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 observa- tions 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 immunode- ficiency 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 CD41 cell numbers in chronically (.2 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 CD81 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 anti- gen than the original PBMC cultures (.10-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