A Comprehensive Strategy for Screening for Xenotransplantation-Relevant Viruses in a Second Isolated Population of Göttingen Minipigs

Abstract

Xenotransplantation using pig tissues and organs is under development in order to alleviate the increasing shortage of human transplants. Since xenotransplantation may be associated with the transmission of porcine microorganisms to the human recipient, the donor pigs should be carefully analyzed, especially for the presence of potentially zoonotic viruses. Göttingen Minipigs (GöMP) are potential donors of islet cells for the treatment of diabetes. Despite the fact that all animals produced at Ellegaard Göttingen Minipigs A/S carry porcine endogenous retroviruses (PERVs) in their genome and that very few animals were infected with porcine cytomegalovirus (PCMV), hepatitis E virus (HEV) and porcine lymphotropic herpesvirus (PLHV), no transmission of these viruses was observed in a preclinical trial transplanting GöMP islet cells into cynomolgus monkeys. Using a new comprehensive strategy, we then analyzed an isolated subpopulation of Göttingen Minipigs which remained at the University of Göttingen. We concentrated on 11 xenotransplantation-relevant viruses and combined co-incubation assays with susceptible human target cells and molecular biological methods to evaluate the risk posed by PERV. All animals in Göttingen carry PERV-A, PERV-B, and PERV-C in their genome but they are not infected with PCMV, PLHV and HEV. The difference may be explained by selection of negative animals and/or de novo infection. The PERV copy number was established using ddPCR (93 copies) and a human-tropic PERV-A/C was found released from PBMCs of one animal with a high expression of PERV-C.

Copy Number and Prevalence of Porcine Endogenous Retroviruses (PERVs) in German Wild Boars

“…Whereas in Göttingen minipigs from Ellegaard no recombinant PERV-A/Cs were found [13], in some Aachen minipigs PERV-A/C was found in the liver and spleen, but not in the peripheral blood mononuclear cells (PBMCs) [29]. In contrast, PERV-A/Cs were found in PBMCs from some Göttingen minipigs bred at the Göttingen university with a high expression of PERVpol and PERV-C [30].…”

Sensitive detection systems for infectious agents in xenotransplantation*

“…The full-length mRNA is commonly expressed in normal tissues, whereas the detection of spliced mRNA is an indicator of PERV expression at a level which allows virus production. 20,38 Viral proteins can be detected by immunofluorescence, immunohistochemistry, and immunoperoxidase assays. Using PERV-specific antibodies, the production of PERV has been shown in PERV-infected 293 cells, pig melanoma cells, or mink cells by immunofluorescence or immunoperoxidase assay.…”

“…37,45 Mitogen treatment has been shown to enhance viral mRNA expression and virus release and is therefore a good tool to increase the sensitivity of these detection methods. 20,37,46 A special form of the RT assay is the product-enhanced reverse transcriptase (PERT) assay, 47 which was also used to detect PERV. 48 Production of PERV particles was shown by electron microscopy (Figure 2), 20,41,42,46 and using PERV-specific antibodies, the specificity has been demonstrated by immune gold electron microscopy.…”

“…20,37,46 A special form of the RT assay is the product-enhanced reverse transcriptase (PERT) assay, 47 which was also used to detect PERV. 48 Production of PERV particles was shown by electron microscopy (Figure 2), 20,41,42,46 and using PERV-specific antibodies, the specificity has been demonstrated by immune gold electron microscopy. 39,40 Infectious virus particles can be detected by infection assays using and the transmembrane envelope protein p15E or on lysates from highly purified virus particles.…”

“…19 Since it is unclear, which porcine microorganisms can be zoonotic in the recipient, some viruses had been selected as xenotransplantation-relevant. 20 Detailed explanations of why HEV, PCMV, PERV, PCV, and some others were included were given in several reviews. [21][22][23][24][25] HEV (genotype 3, gt3) is a well-known zoonotic virus.…”