Introduction: PERVs are integrated in the genome of pigs. PERV-A and PERV-B are present in all pigs, they produce virus particles able to infect human cells (human-tropic) and pose therefore a risk for xenotransplantation. PERV-C is present in most, but not all pigs and it infects only pig cells. In addition, recombinants between PERV-A and PERV-C have been described, which infect human cells and which are characterized by higher replications rates compared with the paternal virus. PERVs are the result of trans-species transmission of precursor retroviruses from different species and further evolution in the pig genome. Despite the ability to infect human cells, non-human primate cells and cells from other species, no PERV transmission was observed in first clinical trials transplanting pig islet cells into diabetic humans, in preclinical trials transplanting cells and organs into non-human primates with remarkably long survival times as well as in infection experiments with numerous animal species.

At present it is unknown how high the probability is that normal pig cells release infectious human-tropic PERV. To study this, we used pig primary peripheral blood mononuclear cells (PBMCs) from 50 pigs including 11 Göttingen Minipigs (GöMP). GöMP will be used in Germany as source of islet cells for the treatment of diabetes in humans.

Methods: Pig PBMCs were stimulated with the T-cell mitogen phytohemagglutinin (PHA), which had been shown in previous studies to increase the expression of PERV mRNA. The stimulated pig PBMCs were co-cultured with human 293 cells shown to be highly susceptible to PERV infections. After several passages, the pig PBMCs died and infection of 293 cells was estimated using a PCR. The virus particles were characterized and the virus genome was sequenced.

Results: Before stimulating the PBMCs, the copy number of PERVs in the genome of GöMP was estimated using ddPCR.  Approximately 78 copies were found, which is slightly higher compared with other pig breeds. After treatment with PHA, PBMCs from only one of the 50 animals, from a GöMP, released PERV able to infect 293 cells. As shown by electron microscopy this virus was a typical gammaretrovirus. This virus was a PERV-A/C recombinant and the break points in the envelope region near the receptor binding domain were different from other PERV-A/C. Passaging on human cells was associated with multimerization of repeats in the long terminal repeat (LTR) carrying transcription factor binding sites. Previously we had shown a similar increase in the length of the LTR and higher virus titers of a PERV-A/C released from PBMCs of another breed. Multimerization of LTR repeats was also observed in the evolution of PERVs.

Conclusion: Release of an infectious human-tropic PERV is a rare event, it happened only in one out of 50 pigs from different breeds and it was a recombinant PERV-A/C. Selection of PERV-C-free animals will prevent such recombination.