Amplification of simian retroviral sequences from human recipients of baboon liver transplants

Abstract

Investigations into the use of baboons as organ donors for human transplant recipients, a procedure called xenotransplantation, have raised the specter of transmitting baboon viruses to humans and possibly establishing new human infectious diseases. Retrospective analysis of tissues from two human transplant recipients with end-stage hepatic disease who died 70 and 27 days after the transplantation of baboon livers revealed the presence of two simian retroviruses of baboon origin, simian foamy virus (SFV) and baboon endogenous virus (BaEV), in multiple tissue compartments. The presence of baboon mitochondrial DNA was also detected in these same tissues, suggesting that xenogeneic "passenger leukocytes" harboring latent or active viral infections had migrated from the xenografts to distant sites within the human recipients. The persistence of SFV and BaEV in human recipients throughout the posttransplant period underscores the potential infectious risks associated with xenotransplantation.

FIG. 1

Immunoblot analysis of serum samples from donor baboons and human transplant recipients. Serum samples from the baboon donors (A) or human transplant recipients (B) were diluted 1:40 and reacted with SFV immunoblot strips. Positive and negative controls were used at a 1:80 dilution. Immunoblotting was performed as previously described with SFV-Bab1 as the antigen. SFV-Bab1 used for making Western blot reagents was isolated from peripheral blood lymphocytes of a baboon that developed lymphoma and has been previously described. Virus was grown in primary human foreskin fibroblasts (HFSM) and harvested during the cytopathic phase of infection. HSFM cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, penicillin (50 U/ml), and streptomycin (50 μg/ml).

FIG. 2

Detection of SFV DNA in tissues from human liver transplant patients. PCR amplification of SFV DNA using LTR primers was performed with DNA from (A) patient 1 and (B) patient 2. Designations are as follows: PBMC, peripheral blood mononuclear cells; LN, lymph node; LIV, liver, KID, kidney; BM, bone marrow; and GI, small intestine. PBMC DNA samples from an SFV-seronegative human and an SFV+ baboon were used as negative and positive controls, respectively. A total of 100 ng of total cellular DNA was added to 200 μM dNTPs, 50 mM KCl, 10 mM Tris, 2.5 mM MgCl₂, bovine serum albumin (BSA, 5 mg/ml), 30 pmol of primer, and 1 U of Taq polymerase (Promega, Madison, WI). Amplification was performed using the following primers: PBF1 (5′-CACTACTCGCTGCGTCGAGAGGTGT-3′) and PBF2 (5′-GGAATTTTGTATATTGATTATCC-3.′). A 329-bp fragment within the SFV LTR (nt 786-1115, based on the HFV sequence) was amplified. Conditions for LTR primers were as follows: 1 cycle at 94°C for 1 min, 52°C for 45 sec and 72°C for 1 min; 1 cycle at 94°C for 1 min, 50°C for 45 sec, and 72°C for 1 min; 1 cycle at 94°C for 1 min, 48°C for 45 sec, and 72°C for 1 min; 1 cycle at 94°C for 1 min, 46°C for 45 sec, and 72°C for 1 min; 25 cycles at 94°C for 1 min, 45°C for 45 sec, and 72°C for 1 min; followed by extension at 72°C for 7 min. Amplified fragments were separated on agarose gels, blotted onto Nytran Plus membranes, and then hybridized with ³²P-labeled SFV-Bab1 LTR probe.

FIG. 3

Unrooted consensus tree of SFV-hu2 LTR DNA from human transplant recipient. The designations for foamy viruses are as follows: SFV-hu2(ln1), -hu2(ln3), -hu2(liv) (human recipient), SFV-1 (macaque), SFV-3, -Agm1 (African green monkey), SFV-Bab1, -Bab3 (baboon), SFV-Gg (gorilla), SFV-6, -7, and CPZ (chimpanzee), SFV-11 (orangutan), SFV-Pat (patas), and SFV-Hm1 (Hamlyn’s guenon). LTR-amplified products were cloned with a TA cloning kit (Invitrogen, San Diego, CA) and sequenced as previously described. Two clones from each of the human samples were randomly selected and purified using a S.N.A.P. miniprep kit (Invitrogen). An Applied Biosysterns (Foster City, CA) automated sequencer model 373A, version 1.2.0, was used to derive the DNA sequences, which were then aligned using the GCG package, version 7.0, and manipulated by hand. Phylogenies were done using 100 bootstrap replicates in generating unrooted consensus trees with PHYLIP version 3.5p. Distances were calculated with Kimura’s two-parameter method (GCG Package version 7.0). Nucleotide sequences have been submitted to GenBank and are available under accession numbers AFO62309, AFO62310, and AFO62311.