Absence of replication-competent human-tropic porcine endogenous retroviruses in the germ line DNA of inbred miniature Swine

Abstract

The potential transmission of porcine endogenous retroviruses (PERVs) has raised concern in the development of porcine xenotransplantation products. Our previous studies have resulted in the identification of animals within a research herd of inbred miniature swine that lack the capacity to transmit PERV to human cells in vitro. In contrast, other animals were capable of PERV transmission. The PERVs that were transmitted to human cells are recombinants between PERV-A and PERV-C in the post-VRA region of the envelope (B. A. Oldmixon, J. C. Wood, T. A. Ericsson, C. A. Wilson, M. E. White-Scharf, G. Andersson, J. L. Greenstein, H. J. Schuurman, and C. Patience, J. Virol. 76:3045-3048, 2002); these viruses we term PERV-A/C. This observation prompted us to determine whether these human-tropic replication-competent (HTRC) PERV-A/C recombinants were present in the genomic DNA of these miniature swine. Genomic DNA libraries were generated from one miniature swine that transmitted HTRC PERV as well as from one miniature swine that did not transmit HTRC PERV. HTRC PERV-A/C proviruses were not identified in the germ line DNAs of these pigs by using genomic mapping. Similarly, although PERV-A loci were identified in both libraries that possessed long env open reading frames, the Env proteins encoded by these loci were nonfunctional according to pseudotype assays. In the absence of a germ line source for HTRC PERV, further studies are warranted to assess the mechanisms by which HTRC PERV can be generated. Once identified, it may prove possible to generate animals with further reduced potential to produce HTRC PERV.

FIG. 1.

Genomic library screening results for transmitting and nontransmitting MS. (A) HTRC-NT MS11852; (B) HTRC-T MS13519. Lambda clones were identified by radioactive hybridization screening prior to PCR and sequencing.

FIG. 2.

Amino acid sequence comparison of the 3′ ends of the env regions of PERV-A clones isolated from both the HTRC-T 13519 (Tr1 to -5) and HTRC-NT 11852 (NTr1 to -4) libraries and PERV-A/C recombinants isolated from 293 cells infected with MS PBMC (t1.2c9 [accession number AF417229], t2a3 [AF417230], t2a5 [AF417231], and t6e5 [AF417232]). T142-derived PERV A/C recombinant sequences are those from the coculture of PBMC from animal 13519 (T142.1 [AY364234] and T142.8 [AY364236]). *, stop codon. Arrows highlight amino acid differences.

FIG. 3.

Genomic DNAs isolated from transmitting and nontransmitting MS and analyzed by PCR for the presence of PERV-A/C recombinant env sequences (top) and pan-PERV env sequences (bottom), using conserved primers. Spiking experiments with recombinant PERV-A/C DNA confirmed the ability of the PCR to detect a single copy of PERV-A/C provirus (not shown). Lanes: 1, PERV-A control (PCR-A60); 2, PERV-B control (PCR-B17); 3, PERV-C env control (PoEV3); 4, MS13519; 5, MS11852; 6, 293 PERV-A/C genomic DNA; 7, 293 cell genomic DNA; M, size marker.

FIG. 4.

Comparison of PERV-A and -B sequences from the GenBank database in the transmembrane region of the envelope. The GenBank accession number for each sequence is indicated at the start of the sequence. The nonrecombinant PERV-A sequence NTr3 (AY28879), which was isolated from MS11852, is included for comparison. Arrow, recombination site; *, stop codon.