Porcine cytomegalovirus (PCMV) infection is widely prevalent among pigs, and PCMV is one of the viruses which may be transmitted during xenotransplantation using pig cells, tissues, or organs. While human cytomegalovirus (HCMV) is a major risk factor for allotransplantation, it is still unclear whether PCMV is able to infect human cells or pose a risk for xenotransplantation. Previously, it was shown that transmission of PCMV after pig kidney to non-human primate transplantations resulted in a significantly reduced survival time of the transplanted organ. To detect PCMV, PCR-based and immunological methods were used. Screening of pigs by Western blot analyses using recombinant viral proteins revealed up to 100% of the tested animals to be infected. When the same method was applied to screen human sera for PCMV-reactive antibodies, positive Western blot results were obtained in butchers and workers in the meat industry as well as in normal blood donors. To exclude an infection of humans with PCMV, the sera were further investigated. PCMV is closely related to human herpesvirus-6 (HHV-6) and human herpesvirus-7 (HHV-7), and a sequence alignment of glycoprotein B suggests that the antibodies may cross-react with identical epitope sequences. HCMV is not related with PCMV, and no correlation between antibody reactivity against PCMV and HCMV was detected. These data indicate that antibodies against PCMV found in humans are cross-reactive antibodies against HHV-6.

Xenotransplantation from pigs could alleviate the shortage of human tissues and organs for transplantation. Means have been identified to overcome hyperacute rejection and acute vascular rejection mechanisms mounted by the recipient. The challenge is to combine multiple genetic modifications to enable normal animal breeding and meet the demand for transplants. We used two methods to colocate xenoprotective transgenes at one locus, sequential targeted transgene placement - 'gene stacking', and cointegration of multiple engineered large vectors - 'combineering', to generate pigs carrying modifications considered necessary to inhibit short to mid-term xenograft rejection. Pigs were generated by serial nuclear transfer and analysed at intermediate stages. Human complement inhibitors CD46, CD55 and CD59 were abundantly expressed in all tissues examined, human HO1 and human A20 were widely expressed. ZFN or CRISPR/Cas9 mediated homozygous GGTA1 and CMAH knockout abolished α-Gal and Neu5Gc epitopes. Cells from multi-transgenic piglets showed complete protection against human complement-mediated lysis, even before GGTA1 knockout. Blockade of endothelial activation reduced TNFα-induced E-selectin expression, IFNγ-induced MHC class-II upregulation and TNFα/cycloheximide caspase induction. Microbial analysis found no PERV-C, PCMV or 13 other infectious agents. These animals are a major advance towards clinical porcine xenotransplantation and demonstrate that livestock engineering has come of age.

Porcine circovirus 2 (PCV2) is the causative agent of a whole series of diseases in pigs, called PCV2 diseases (PCVD). The most relevant of them is the systemic disease (PCV2-SD), formerly called post weaning multi systemic wasting syndrome (PMWS). Xenotransplantation using pig cells, tissues and organs is under development to overcome the shortage of human transplants for the treatment of tissue and organ failure. Xenotransplantation requires functional cells, tissues or organs from healthy animals and therefore the donor pigs should be free of PCV2. Selection of PCV2-free animals will also prevent transmission of the virus to the human recipient. Using a PCR method, (i) Göttingen Minipigs, which are well characterised and which were already used in pig to non-human primate xenotransplantations, (ii) newly generated Aachen Minipigs, (iii) genetically modified pigs generated for xenotransplantation, (iv) pigs from a slaughterhouse and (v) pigs from a German farm were screened for PCV2. 50% of the Aachen minipigs and 14% of Göttingen minipigs were PCV2 positive, but the animals were apparently healthy. None of the slaughterhouse animals, the farm animals and the genetically modified animals were positive for PCV2, because they had been vaccinated. The data indicate that PCV2 may be found in healthy pigs even under SPF conditions, and that vaccination is a powerful tool to prevent infection.

Background: To prevent transmission of zoonotic microorganisms from pig transplants to human recipients when performing xenotransplantation using pig cells, tissues, or organs, donor pigs have to be carefully characterized. Gottingen minipigs (GoMP) are often used for various biomedical investigations and are well characterized concerning the presence of numerous bacteria, fungi, viruses, and parasites. Recently, we studied the prevalence and expression of porcine endogenous retroviruses and the prevalence of hepatitis E virus (HEV) in GoMP. Here, we studied the presence of the porcine cytomegalovirus (PCMV) and porcine lymphotropic herpesviruses (PLHV) and extended testing for hepatitis E virus (HEV).Methods: PCR, nested PCR, real-time PCR, real-time RT-PCR, and Western blot analyses were used to estimate the prevalence of PCMV, PLHV-1, PLHV-2, PLHV-3, and HEV.Results: Using different PCR methods, and different source materials, PCMV was found in 10 of 26 adult GoMP, which had been derived originally by cesarean section and kept under specified pathogen-free conditions. Only highly sensitive methods gave positive results, not methods of lower sensitivity. The virus load in all positive animals was low (< 100-200 copies per mL). PLHV-1, PLHV-2, and PLHV-3 were not detected by PCR; however, an anti-PLHV immune response was found in one of 10 animals tested by Western blot analyses. HEV was detected by RT-PCR in two of nine tested animals, but no anti-HEV immune response was observed.Conclusion: Using highly sensitive methods, PCMV, HEV, and PLHV were found in some GoMP, suggesting that these viruses may be introduced through the placenta. The results show that highly sensitive methods are required to characterize pigs to be used for xenotransplantation to prevent virus transmission.

BACKGROUND:After transplantation of pig hearts into baboons, a particularly high increase of liver parameters was observed in 1 animal. To evaluate whether porcine hepatitis E virus (HEV) was involved in the pathological changes, the donor pig and the recipient baboon were screened for the presence of HEV.MATERIAL AND METHODS:Screening for HEV was performed using highly sensitive and specific PCR methods as well as immunological screening for HEV-specific antibodies.RESULTS:HEV was not detected in the donor pig or the baboon recipient. At necropsy, histopathological examination of liver sections showed acute coagulative necrosis of hepatocytes and hemorrhage, but minimal inflammatory cell activity.CONCLUSIONS:The liver failure observed in the recipient animal was not due to transmission of porcine HEV. Liver failure could have been caused by the onset of cardiac failure related to delayed transplant rejection.

Porcine cytomegalovirus (PCMV) may be harmful for human recipients if xenotransplantation using pig cell, tissue or organ will be performed transmitting the virus from donor pigs to human recipients. PCMV is widespread in pigs and closely related to human pathogenic herpesviruses, however there are no data concerning infection of humans. In contrast, recently it had been shown that transplantation of organs from pigs infected with PCMV into non-human primate recipients resulted in a significant reduction of the survival time compared with the transplantation of organs from uninfected pigs. To prevent transmission of PCMV in future pig to human xenotransplantations, sensitive and specific detection methods should be used. Here a new Western blot assay using recombinant proteins corresponding to two domains of the glycoprotein gB of PCMV is described. With this assay, the presence of PCMV-specific antibodies in different pig breeds was analysed. Antibodies were detected in a high percentage of animals, in one breed up to 85%.

BackgroundTo alleviate the shortage of human donor organs or tissues for the treatment of organ and tissue failure including diabetes, pigs are considered suitable donor animals. As organs from conventional pigs are usually too large, those from minipigs may be better suited. We recently characterized the Gottingen Minipigs, a breed well characterized concerning the presence of zoonotic microorganisms and found hepatitis E virus (HEV) and porcine cytomegalovirus (PCMV) in some animals. Here, we characterize another minipig, the Aachen Minipig (AaMP), a pig breed recently established close to the town Aachen in Germany.MethodsThe animals were tested for the prevalence and expression of porcine endogenous retroviruses (PERVs) and the presence of some selected microorganisms, among them HEV, PCMV, and porcine lymphotropic herpesviruses (PLHVs) using highly sensitive and specific PCR and RT-PCR methods. In addition, we screened for antibodies against HEV and PLHV.ResultsPERV-A, PERV-B, and PERV-C sequences were found in the genome of all Aachen Minipigs. HEV RNA was found by real-time RT-PCR in most, and DNA of PCMV, PLHV-2, and PLHV-3 was found by PCR in some animals. The animals were free of eight other microorganisms tested, but some were seropositive for porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine epidemic diarrhea virus (PEDV).ConclusionBased on medical examinations by veterinarians, the AaMP are in a good health status and seem to harbor only few microorganisms. To improve their status for use as donor pigs in xenotransplantation, the viruses detected might be eliminated by selection of negative animals, Cesarean section, and vaccination.

Porcine lymphotropic herpesviruses (PLHV-1, -2, and -3) are widespread in pigs and closely related to the human pathogenic gammaherpesviruses Epstein-Barr virus (human herpesvirus 4, HHV-4) and Kaposi sarcoma herpesvirus (HHV-8). In minipigs, PLHV-1 causes a porcine post-transplantation lymphoproliferative disorder (PTLD) after experimental transplantations. Porcine PTLD comes with clinical symptoms similar to those of human PTLD, a serious complication of solid organ and allogeneic bone marrow transplantation linked to HHV-4. Since PLHVs may be transmitted from donor pigs to the human recipient of xenotransplants (pig cells, tissues or organs), sensitive and specific methods should be developed to detect and eliminate PLHVs. Here we describe an ELISA and a Western blot assay using recombinant glycoprotein B of PLHV-1. Using both assays, the presence of specific antibodies in different pig breeds as well as in German slaughterhouse workers was analysed. Antibodies were detected in some animals, but not in human subjects.

Transplantation of pig islet cells for the treatment of diabetes may be a more effective approach compared with the application of insulin. However, before introduction into the clinic, efficacy and safety of this treatment have to be shown. Non-human primate models may be used for this, despite the fact that they are characterised by several limitations. Here we investigate the prevalence of porcine endogenous retroviruses (PERVs), which are present in the genome of all pigs and which may infect human cells, as well as of porcine herpes viruses in donor pigs and their potential transmission to non-human primate recipients. Despite the fact that all three subtypes of PERV were present in all and porcine cytomegalovirus (PCMV) was found in some of the pigs, neither PERVs nor PCMV were found in the recipient animals under the experimental conditions applied. Porcine lymphotropic herpes viruses (PLHV) were not found in the donor pigs, hepatitis E virus (HEV) was not found in the recipients.

Xenotransplantation using porcine cells, tissue or organs may be associated with transmission of various pathogens that can induce zoonosis. Highly specific and sensitive methods detecting porcine endogenous retroviruses (PERVs), hepatitis E virus (HEV), porcine cytomegalovirus (PCMV) were developed and used to screen (i) wild-type Gottingen Minipigs, a breed produced under specified pathogen-free (spf) conditions, (ii) wild-type Aachen Minipigs, a newly created pig breed in Germany, and (iii) pigs produced carrying various transgenic modifications to prevent transplant rejection were analysed. Moreover, some animals were tested for the presence of >49 other microorganisms, incl. porcine lymphotropic herpes viruses (PLHV). Human-tropic PERV-A and PERV-B were found in all pigs und PERV-C was detected in all tested Gottingen Minipigs and in more than 90% of the other pigs. Although PERV-C does not infect human cells, it may represent a special risk when it recombines with PERV-A, as high-titre PERV-A/C were able to infect human cells in vitro. The expression level of PERV in all examined animals, as analysed in a mitogen stimulation assay, was found to same low range as previously reported for German landrace pigs. HEV was found in 27% of the Gottingen Minipigs, the prevalence of the herpes viruses was below 10%. Immunological methods based on recombinant viral proteins were developed to screen for antibodies against PERV, HEV, PCMV and PLHV. Antibodies against HEV were found only in few animals, whereby in piglets they were of maternal origin. The presence of HEV in sera of three sows six days after parturition and in their offspring in a well characterised SPF facility suggests vertical transmission of the virus. In contrast to PERVs, which are integrated in the porcine genome, HEV and herpes viruses can be eliminated using Caesarean section and eventually antiviral treatment. Most importantly, when islet cells from non-transgenic and transgenic pigs expressing immunomodulatory molecules were transplanted into marmosets, no transmission of PERV, HEV, PCMV and PLHV was observed, although four out of 14 donor pigs were PCMV positive. No transmission of PERV, HEV, PCMV and PLHV was observed after hearts from triple transgenic pigs were transplanted into baboons. These data indicate that sensitive and reliable methods for the detection of pathogens are available and that screening of putative donor pigs of porcine xenotransplants is critical to ensure microbiological safety in clinical xenotransplantation. Supported by the German Research Foundation (DFG) Transregio Collaborative Research Centre 127.