Identification of exogenous forms of human-tropic porcine endogenous retrovirus in miniature Swine

Abstract

The replication of porcine endogenous retrovirus subgroup A (PERV-A) and PERV-B in certain human cell lines indicates that PERV may pose an infectious risk in clinical xenotransplantation. We have previously reported that human-tropic PERVs isolated from infected human cells following cocultivation with miniature swine peripheral blood mononuclear cells (PBMC) are recombinants of PERV-A with PERV-C. Here, we report that these recombinants are exogenous viruses in miniature swine; i.e., they are not present in the germ line DNA. These viruses were invariably present in miniature swine that transmitted PERV to human cells and were also identified in some miniature swine that lacked this ability. These data, together with the demonstration of the absence of both replication-competent PERV-A and recombinant PERV-A/C loci in the genome of miniature swine (L. Scobie, S. Taylor, J. C. Wood, K. M. Suling, G. Quinn, C. Patience, H.-J. Schuurman, and D. E. Onions, J. Virol. 78:2502-2509, 2004), indicate that exogenous PERV is the principal source of human-tropic virus in these animals. Interestingly, strong expression of PERV-C in PBMC correlated with an ability of the PBMC to transmit PERV-A/C recombinants in vitro, indicating that PERV-C may be an important factor affecting the production of human-tropic PERV. In light of these observations, the safety of clinical xenotransplantation from miniature swine will be most enhanced by the utilization of source animals that do not transmit PERV to either human or porcine cells. Such animals were identified within the miniature swine herd and may further enhance the safety of clinical xenotransplantation.

Figures

FIG. 1.

Schematic showing the matings that were initiated to investigate the incidence of PERV transmission in vitro. Human-tropic transmitting animals are indicated with dark fill; nontransmitting animals are indicated with light fill. Note all parental animals produced PERV that infected pig cells in vitro. The transmission phenotypes of the offspring animals are not indicated but are presented in Tables 1 to 3.

FIG. 2.

PERV-A/C recombinants can be identified in the PBMC of miniature swine and are transmitted to 293 cells. (A) PCR and RT-PCR assays specific for the detection of PERV-A/C recombinant sequences were performed on miniature swine PBMC and PERV-infected 293 cells with primer pair 1 (see Materials and Methods). Representative samples from a transmitting animal and the associated in vitro 293 cell transmission assay are presented. Lanes: 1, PBMC RT-PCR; 2, PBMC PCR; 3, 293 cell RT-PCR; 4, 293 cell PCR; M, marker. (B) Determination of the sensitivity of the PERV-A/C PCR, indicating that PERV-A/C recombinant viruses are not endogenous. PERV-A/C plasmids were spiked into approximately 5,000 cell equivalents (100 ng) of porcine DNA at the copy number per cell equivalent indicated.

FIG. 3.

Fluorescence in situ hybridization for PERV-C RNA expression. Strong expression of PERV-C can be detected in a minority of PBMC from transmitting, but not nontransmitting, miniature swine. Miniature swine PBMC were analyzed by in situ hybridization for PERV RNA expression with a probe for PERV-C envelope sequences.

FIG. 4.

PERV-null animals produce significantly less virus that transmitting and nontransmitting miniature swine. PBMC were stimulated with PHA for 72 h, at which point the supernatant was tested for RT activity and the PBMC were used in transmission assays.