No 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.

Xenotransplantation using pig organs has achieved survival times up to 195 days in pig orthotopic heart transplantation into baboons. Here we demonstrate that in addition to an improved immunosuppressive regimen, non-ischaemic preservation with continuous perfusion and control of post-transplantation growth of the transplant, prevention of transmission of the porcine cytomegalovirus (PCMV) plays an important role in achieving long survival times. For the first time we demonstrate that PCMV transmission in orthotopic pig heart xenotransplantation was associated with a reduced survival time of the transplant and increased levels of IL-6 and TNFα were found in the transplanted baboon. Furthermore, high levels of tPA-PAI-1 complexes were found, suggesting a complete loss of the pro-fibrinolytic properties of the endothelial cells. These data show that PCMV has an important impact on transplant survival and call for elimination of PCMV from donor pigs.

Porcine circovirus type 2 (PCV2) is associated with several syndromes in growing pigs, including postweaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome. In the present study, a previously undescribed neurovascular disorder associated with a PCV2 infection is described. Sixteen pigs showed clinical signs of wasting and neurologic deficits. Acute hemorrhages and edema of cerebellar meninges and parenchyma due to a necrotizing vasculitis resulted in degeneration and necrosis of the gray and white matter. Few to numerous PCV2 DNA and antigen-bearing endothelial cells were detected in affected areas of the brain using in situ hybridization and immunohistochemistry. Conventional histochemical stains, as well as the detection of caspase 3 activity and DNA strand breaks by the terminal transferase dUTP nick end labeling assay, showed numerous apoptotic endothelial cells in the vascular lesions observed. Sequencing of various brain-derived PCV2-specific amplicons revealed a strong identity between different isolates and an 89 to 100% identity to previous isolates. The phylogenetic tree showed that there was no clustering of isolates correlating to clinical signs or geographic distribution. This previously undescribed PCV2-associated neurologic disease has features of both postweaning multisystemic wasting syndrome and, to a lesser extent, porcine dermatitis and nephropathy syndrome. The available evidence suggests that direct virus-induced apoptosis of endothelial cells plays a role in the pathogenesis of this unusual PCV2-associated cerebellar vasculitis.

Porcine circovirus 3 (PCV3) is a newly described member of the virus family Circoviridae. PCV3 is highly distributed among pigs and wild boars worldwide. A sudden introduction of PCV3 was recently observed in a herd of triple genetically modified pigs generated for xenotransplantation. These animals were used as donor pigs for orthotopic heart transplantation into baboons. In four cases, PCV3-positive hearts were transplanted, and transmission of PCV3 to the recipient was observed. PCV3 was found in all organs of the recipient baboons and a higher virus load was found in animals with a longer survival time of the transplant, indicating replication of the virus. This is the first report showing trans-species transmission of PCV3 to baboons by transplantation of a heart from a PCV3-positive donor pig. Sequence analysis showed that PCV3a and PCV3b were present in the infected pigs and were transmitted. Experiments to infect human 293 cells with PCV3 failed.