Microchimerism is the presence of cells in an individual that have originated from another individual. The most common form of microchimerism is fetomaternal microchimerism, i.e., cells from a fetus pass through the placenta and establish cell lineages within the mother. Microchimerism was also described after transplantation of human organs in human recipients. Consequently, microchimerism may also be expected in xenotransplantation using pig cells or organs. Indeed, microchimerism was described in patients after xenotransplantations as well as in non-human primates after transplantation of pig organs. Here for the first time a comprehensive review of microchimerism in xenotransplantation is given. Since pig cells contain porcine endogenous retroviruses (PERVs) in their genome, detection of proviral DNA in the transplant recipients may be misinterpreted as infection of the recipient with PERV. To prevent this, methods discriminating between infection and microchimerism are described. This knowledge will be important for the interpretation of screening results in forthcoming human xenotransplantations.

Auckland Island pigs represent an inbred population of feral pigs isolated on the subantarctic island for over 100 years. The animals have been maintained under pathogen-free conditions in New Zealand, they are well characterized virologically and have been used as donor sources in first clinical trials of porcine neonatal islet cell transplantation for the treatment of human diabetes patients. The animals do not carry any of the xenotransplantation-relevant viruses and in the first clinical trials no porcine viruses including porcine endogenous retroviruses (PERVs) have been transmitted to the human recipients. PERVs pose a special risk in xenotransplantation since they are part of the pig genome. When the copy number of PERVs in these animals was analyzed using droplet digital PCR and primers binding to a conserved region of the polymerase gene (PERVpol), a copy number typical for Western pigs was found, confirming previous phylogenetic analyses of microsatellites and mitochondrial analyses showing a closer relationship to European pigs than to Chinese pigs. When kidney cells from very young piglets were analyzed, only around 20 PERVpol copies were detected. Using these cells as donors in somatic cell nuclear transfer (SCNT), animals were born showing a PERVpol copy numbers between 35 and 56. This data indicates that Auckland Is-land pigs have a similar copy number in comparison with other Western pig breeds and that the copy number is higher in adult animals compared with cells from young piglets. Most im-portantly, PERV-C free animals were selected and the absence of additional eight porcine viruses was demonstrated.

Auckland Island pigs represent an inbred population of feral pigs isolated on the subantarctic island for over 100 years. The animals have been maintained under pathogen-free conditions in New Zealand, they are well characterized virologically and have been used as donor sources in first clinical trials of porcine neonatal islet cell transplantation for the treatment of human dia-betes patients. The animals do not carry any of the xenotransplantation-relevant viruses and in the first clinical trials no porcine viruses including porcine endogenous retroviruses (PERVs) have been transmitted to the human recipients. PERVs pose a special risk in xenotransplantation since they are part of the pig genome. When the copy number of PERVs in these animals was analyzed using droplet digital PCR, a copy number typical for Western pigs was found, confirming previous phylogenetic analyses of microsatellites and mitochondrial analyses showing a closer relationship to European pigs than to Chinese pigs. When kidney cells from very young piglets were analyzed, only around 20 PERVpol copies were detected. Using these cells as nuclear donors in a somatic cell nuclear transfer (SCNT), animals were born showing PERVpol copy numbers between 35 and 56. This data indicates that Auckland Island pigs have a lower copy number in comparison with other Western pig breeds and that the copy number is higher in adult animals compared with cells from young piglets. Most importantly, PERV-C free animals were selected and the absence of additional eight porcine viruses was demonstrated.

The successful advancement of xenotransplantation has led to the development of highly sensitive detection systems for the screening for potentially zoonotic viruses in donor pigs and preventing their transmission to the recipient. To validate these methods, genetically modified pigs generated for xenotransplantation, numerous minipig and other pig breeds have been tested, thereby increasing our knowledge concerning the pig virome and the distribution of pig viruses. Of particular importance are the porcine cytomegalovirus, a porcine roseolovirus (PCMV/PRV) and the hepatitis E virus genotype 3 (HEV3). PCMV/PRV has been shown to reduce the survival time of pig transplants in non-human primates and was also transmitted in the first pig heart transplantation to a human patient. Here, we determined the sensitivities of our methods to detect PCMV/PRV, HEV3, porcine lymphotropic herpesvirus - 1 (PLHV-1), PLHV-2, PLHV-3, porcine circovirus 2 (PCV2), PCV3, PCV4 and porcine parvovirus 1 (PPV1) efficiently and used the methods to screen indigenous Greek black pigs. Since porcine endogenous retroviruses (PERVs) of type A and B are integrated in the genome of all pigs, but PERV-C is not, the animals were screened for PERV-C and PERV-A/C. Our detection methods were sensitive and detected PCMV/PRV, PLHV-1, PLHV-1, PLHV-3, PVC3 and PERV-C in most animals. PPV1, HEV3, PCV4 and PERV-A/C were not detected. These data are of great interest since the animals are healthy and very resistant to diseases.

Xenotransplantation is, like allotransplantation, usually associated with microchimerism, e.g., the presence of cells from the donor in the recipient. Microchimerism was reported in first xenotransplantation trials in humans as well as in most preclinical trials in non-human primates (for review see Denner, Viruses 2023, 15, 190). When using pigs as xenotransplantation donors, their cells contain porcine endogenous retroviruses (PERVs) in their genome. That makes it difficult to discriminate between microchimerism and PERV infection of the recipient. Here, we demonstrate which virological methods should be used to identify microchimerism, first of all screening for porcine cellular genes. Using porcine short interspersed nuclear sequences (SINES), which have hundred thousands of copies in the pig genome significantly increased the sensitivity of the screening for pig cells. Second, absence of PERV RNA demonstrated an absence of viral genomic RNA or expression as mRNA. Finally, absence of antibodies against PERV proteins conclusively demonstrated an absence of a PERV infection. When applying these methods for analyzing baboons after pig heart transplantation, microchimerism could be demonstrated in all animals. These methods can be used in future clinical trials.