Sensitive detection systems for infectious agents in xenotransplantation.

Xenotransplantation of pig cells, tissues, or organs may be associated with transmission of porcine microorganisms, first of all of viruses, to the transplant recipient, potentially inducing a disease (zoonosis). I would like to define detection systems as the complex of sample generation, sample preparation, sample origin, time of sampling, and the necessary negative and positive controls along with the specific detection methods, either PCR-based, cell-based, or immunological methods. Some xenotransplantation-relevant viruses have already been defined; others are still unknown. The PCR-based methods include PCR and real-time PCR for DNA viruses, and RT-PCR and real-time RT-PCR for RNA viruses as well as for virus expression studies at the RNA level. Furthermore, droplet digital PCR (ddPCR) can be used for the determination of virus and provirus copies. To detect expression at the protein level, immunofluorescence, immunohistochemistry, and Western blot analyses can be used. To detect virus production and to detect infectious viruses, electron microscopy and infection assays can be used. Furthermore, immunological methods such as Western blot analysis or ELISA can be used to detect virus-specific antibodies. Detection of antiviral antibodies is a reliable and sensitive indirect detection method. For these immunological methods, purified viruses, recombinant viral proteins, or synthetic peptides are used as antigens and control sera and control antigens are needed. All these methods have been used in the past for the characterization of different pig breeds including genetically modified pigs generated for xenotransplantation and for the screening of recipients in preclinical and clinical xenotransplantations. Whereas in preclinical trials a few porcine viruses have been transmitted to the non-human primate recipients, in first clinical trials no such transmissions to humans were observed. Further improvement of the detection systems and their application in virus elimination programs will lead to clean donor animals and a safe xenotransplantation.