Comparative Medicine Vol 62, No 1 Copyright 2012 February 2012 by the American Association for Laboratory Animal Science Pages 61–68 Original Research Effects of Simian Betaretrovirus Serotype 1 (SRV1) Infection on the Differentiation of Hematopoietic Progenitor Cells (CD34+) Derived from Bone Marrow of Rhesus Macaques (Macaca mulatta) Nestor A Montiel,1,2,* Patricia A Todd,2 JoAnn Yee,2 and Nicholas W Lerche2 Peripheral blood cytopenias, particularly persistent anemia and neutropenia, are commonly associated with simian betaretrovirus infection of Asian monkeys of the genus Macaca. The pathogenetic mechanisms underlying these hematologic abnormalities are not well understood. The current study investigated the in vitro tropism of simian betaretrovirus (SRV) for both hematopoietic progenitor (CD34+) and stromal cells obtained from rhesus macaque bone marrow and assessed the effects of infection on hemato- poietic progenitor cell differentiation in vitro. After in vitro exposure, SRV proviral DNA could be demonstrated by real-time PCR in cells and the reverse transcriptase assay in supernatants from SRV-exposed progenitor-associated stroma, but not in differentiated colonies derived from SRV-exposed progenitors. Furthermore, in vitro exposure involving cell–cell contact of uninfected CD34+ progenitor cells with SRV-infected stromal cells resulted in a statistically significant reduction in granulocyte–macrophage colony formation in absence of detectable SRV-infection of progenitor cells. Reduction in colony formation occurred in a ‘dose-dependent’ fashion with increasing contact time. No effects on erythroid lineages and RBC differentiation were noted. Our results suggest that hematologic abnormalities observed during SRV disease (natural or experimental) of rhesus macaques may not result from direct effects of viral infection of progenitor cell populations, but rather be (at least in part) a consequence of SRV infection of supportive bone marrow stroma with secondary effects on differentiation of associated progenitor cells. Abbreviations: E, erythroid; GEMM, granulocytic–erythroid–monocytic–megakaryocytic; GM, granulocytic–macrophage; rh, recombinant human; SRV1, simian type D retrovirus 1. Simian betaretrovirus (formerly simian type D retrovirus; SRV) naturally acquired SRV infection of macaques20,37 and are general- comprises a group of closely related exogenous retroviruses for ly more pronounced in animals with active viremia than in persis- which the natural hosts are Asian monkeys of the genus Maca- tently infected but nonviremic animals.19 Bone marrow changes ca. SRV is now classified in the genus Betaretrovirus subfamily accompanying peripheral cytopenias have been described as Orthoretrovirinae and is the etiologic agent of an immunosup- hypercellular in early stages becoming hypocellular only in the pressive syndrome in various species of macaques used in bio- late stages of SRV infection.37 The specific mechanisms respon- medical research.9,15,27,36,38,39,42 SRV exhibits a broad cellular tropism sible for the origin SRV-associated hematologic abnormalities are for cells of both lymphoid and nonlymphoid tissues.40 Clinical not known, and relevant studies of this phenomenon are few. By and pathologic manifestations of SRV infection range from sub- analogy with other viral infection models exhibiting hematologic clinical (carrier state) to fatal immunosuppressive disease.20,30 SRV abnormalities, a variety of underlying pathogenetic mechanisms is an important pathogen in macaques maintained for use in bio- acting at the bone marrow level may contribute to peripheral cy- medical research, and undetected infections represent a potential topenias, including virus-induced dysregulation of cytokine or confounding variable in research protocols. As a result, SRV is chemokine production, production of soluble factors that inhibit one of several persistent viruses targeted for elimination in SPF normal hematopoiesis, direct viral infection of hematopoietic colony development.28,31 progenitor cells that leads to altered function and metabolism, Hematologic abnormalities, particularly anemia and neutro- and infection and alteration of cells comprising the bone mar- penia, are common laboratory findings in both experimental and row microenvironment that could indirectly impair the ability of progenitors to differentiate into lineage-committed cells.34,35,46,54 Anemia and leukopenias associated with reduced progenitor Received: 16 May 2011. Revision requested: 28 Jun 2011. Accepted: 07 Sep 2011. cell proliferation (by either direct susceptibility of progenitors to 1Center for Comparative Medicine and 2California National Primate Research Center, University of California, Davis, California. infection or indirect effects of the infected microenvironment) and *Corresponding author. Email: [email protected] impaired iron utilization have been observed in several diseases 61 Vol 62, No 1 Comparative Medicine February 2012 of viral origin, including simian parvovirus of macaques,44 hu- Suspension cultures of hematopoietic progenitor cells were man parvovirus B19 infection,6,43 simian and human immunode- maintained for either 7 or 20 d at 3 × 105 cells/mL in 16-mm wells ficiency syndromes,12,26,46,51,54 and feline retroviral infections.23,35,47,53 in either 10% FBS in Iscove-modified Dulbecco medium (Gibco) Limited studies of SRV-associated cytopenias have suggested that or in serum-free expansion medium (StemSpan SFEM, StemCell adverse hematologic effects of SRV infection may originate at the Technologies) containing recombinant human (rh) cytokines IL3 bone marrow level.37,42 The objectives of the current study were to (20 ng/mL), IL6 (20 ng/mL), SCF (100 ng/mL), and Flt3 ligand determine 1) the in vitro tropism of SRV1 for both CD34+ hema- (100 ng/mL; StemCell Technologies). Long-term stromal cell cul- topoietic progenitors and supportive stroma cell components of tures were maintained for 4 to 8 wk in either 35- or 16-mm wells rhesus macaque bone marrow and 2) the effects of SRV infection in 20% FBS in DMEM (Gibco) containing 10-6 M hydrocortisone of either or both marrow constituent cell populations on in vitro (StemCell Technologies). Primary stromal cells were obtained differentiation of erythrocytic and granulocytic precursor cells. from the CD34+ retention column wash fractions (CD34–). Stromal cells were counted, seeded onto 9.5- or 1.91-mm2 wells (35- and Materials and Methods 16-mm dishes, respectively) in 20% FBS in DMEM (Gibco) con- taining 10-6 M hydrocortisone (StemCell Technologies) at initial Animals and bone marrow collection. Twelve healthy adult concentrations of 5 × 105 or 3 × 105 cells/mL, and incubated at 37 rhesus macaques (Macaca mulatta) maintained as blood donors °C in humidified 5% CO until layers were completely confluent. and representative of the normal adult rhesus population at our 2 Stromal cell cultures were maintained for a maximum of 12 wk. institution (California National Primate Research Center, Univer- Infection of progenitor and stromal cells with SRV1. Infection of sity of California, Davis, California) were used as bone marrow rhesus bone marrow CD34+ hematopoietic progenitor cells with donors for these experiments. One rhesus macaque (MMU 22396) chronically infected with SRV1 (confirmed by PCR and serology) SRV1 was performed by exposure of progenitor cells to infec- but without clinical signs of SRV disease served as a donor of tious supernatant obtained from selected SRV1-infected Raji cell bone marrow cells and was used as a positive control for some cultures presenting high reverse transcriptase activity (greater assays during the study. All donors (except MMU 22396) were than 20-fold background). Reverse transcriptase activity was de- determined to be free of SRV infection by serology and RT-PCR. termined by using a commercially available detection kit (Cavidi Our facility is fully AAALAC-accredited, and all animals were Tech AB, Uppsala, Sweden). The multiplicity of infection used housed and handled in accordance with the Animal Welfare ranged from 0.1 to 1. After 1- or 4-h incubation at 37 °C, with Act and the Guide for the Care and Use of Laboratory Animals.24 All equal volumes of infected or uninfected cell supernatant, cells procedures associated with this research were approved by the were resuspended in PBS (Gibco) and seeded onto culture dish- IACUC of the University of California, Davis. Macaques were es in semisolid medium to assay progenitor cell differentiation 5 anesthetized with ketamine HCl (10 mg/kg IM; Vetamine, Scher- as previously described. Semiconfluent stromal cultures were ing-Plough, Kenilworth, NJ). The bone marrow extraction site incubated twice (with a 24-h interval between exposures) with was prepared aseptically, and 0.5 mL lidocaine (Abbott Laborato- SRV-infected cell-free supernatant for 4 h at 37 °C each time, at ries, Alameda, CA) was administered subcutaneously. Bone mar- an estimated multiplicity of infection of 0.5. After the second in- row (5 to 10 mL) was aspirated from either the humerus or iliac cubation, cells were washed twice in PBS (Gibco) and then resus- −6 crest of each animal by using a heparin-coated (0.1 mL) syringe pended in 10% FBS in DMEM containing 10 M hydrocortisone with a 20-gauge, 1.5-in. spinal needle (Becton Dickinson, San (StemCell Technologies) for long-term maintenance. Jose, CA). A 50-µL aliquot of each sample was used to determine Progenitor cell differentiation with recombinant cytokines. the nucleated cell