Porcine endogenous retroviruses (PERVs) infect human cells in vitro and therefore represent a risk for xenotransplantation. However, first clinical transplantations of pig cells into humans or ex ...vivo perfusions did not result in transmission of PERVs. On the other hand, recent experiments with SCID mice demonstrated infections with PERV in vivo. In order to define and characterize human target cells, we studied numerous primary human cells and cell lines. Infection with PERVs was shown for human peripheral blood mononuclear cells, primary endothelial cells, and primary aortic smooth muscle cells as well as lymphocytic, monocytic, and epithelial cell lines.
Robert Koch-Institut, Nordufer 20, D-13353 Berlin, Germany 1
Paul-Ehrlich-Institut, Paul-Ehrlich-Strasse 51-59, D-63225 Langen, Germany 2
Author for correspondence: Joachim Denner at Robert ...Koch-Institut. Fax +49 30 4547 2801. e-mail dennerj{at}rki.de
Using transgenic pigs as the source of cells or organs for xenotransplantation is associated with the risk of porcine endogenous retrovirus (PERV) transmission. Multiple proviruses are integrated into the genome of all pigs, and virus particles, some of which are able to infect human cells, are released from normal pig cells. In order to evaluate the potential risk posed by the transmission of PERVs, in vitro infection studies were performed as a basis for small animal as well as non-human primate models. In vitro infectivity was demonstrated for permanent cell lines and primary cells from a wide range of species. Productive infection was shown using reverse transcriptase (RT) assays and RTPCR for mink, feline and human kidney cell lines, primary rhesus peripheral blood mononuclear cells (PBMCs), and baboon spleen cells and PBMCs as well as for different human lymphoid and monocyte cell lines and PBMCs. In an attempt to establish a small animal model, naive guinea pigs, non-immunosuppressed rats, rats immunosuppressed by cyclosporin-A and immunosuppressed rats treated with cobra venom factor were inoculated with PERVs produced from porcine kidney PK-15 cells, infected human 293 kidney cells and mitogen-stimulated porcine PBMCs. Animals were also inoculated with PERV-producing PK-15 and 293 cells. No antibodies against PERV and no provirus integration were observed in any of the treated animals. This suggests that productive infection of these animals did not occur in this experimental setting.
For xenotransplantation, the transplantation of animal cells, tissues and organs into human recipients, to date, pigs are favored as potential donors. Beside ethical, immunological, physiological and ...technical problems, the microbiological safety of the xenograft has to be guaranteed. It will be possible to eliminate all of the known porcine microorgansims in the nearby future by vaccinating or specified pathogen-free breeding. Thus, the main risk will come from the porcine endogenous retroviruses (PERVs) which are present in the pig genome as proviruses of different subtypes. PERVs will therefore be transmitted, with the xenograft, to the human recipient. PERVs can infect numerous different types of human primary cells and cell lines in vitro and were shown to adapt to these cells by serial passaging on uninfected cells. Furthermore, PERVs have high homology to other retroviruses, such as feline leukemia virus (FeLV) or murine leukemia virus (MuLV), which are known to induce tumors or immunodeficiencies in the infected host. To evaluate the potential risk of a trans-species transmission of PERV in vivo, naive and immunosuppressed rats, guinea pigs and minks were inoculated with PERV and screened over a period of 3 months for an antibody reaction against PERV proteins or for the integration of proviral DNA into the genomic DNA of the host's cells. Furthermore, we inoculated three different species of non-human primates, rhesus monkey (
Macaca mulatta), pig-tailed monkey (
Macaca nemestrina) and baboon (
Papio hamadryas) with high titers of a human-adapted PERV. To simulate a situation in xenotransplantation, the animals received a daily triple immunosuppression using cyclosporine A, methylprednisolone and RAD, a rapamycin derivative, presently under development by Novartis. None of the small laboratory animals or the non-human primates showed production of antibodies against PERV or evidence of integration of proviral DNA in blood cells or cells of several organs, 3 months after virus inoculation, despite the observation that cells of the animals used in the experiment were infectible in vitro. This apparent difference in the outcome of the in vitro and the in vivo data might be explained by an efficient elimination of the virus by the innate or adaptive immunity of the animals.
Porcine endogenous retroviruses (PERVs) are considered a special risk for xenotransplantation because they are an integral part of the porcine genome and are able to infect cells of numerous species ...including humans in vitro. Among these cells, the mink lung epithelial cell line Mv1Lu could be productively infected with PERV. Provirus integration was detected by PCR, expression of viral proteins was shown by immunostaining and reverse transcriptase was detected in cell supernatants. PERV produced from mink cells could infect both, uninfected mink Mv1Lu cells and uninfected human 293 cells, with considerably higher virus production by human cells. Typical type C retroviruses were observed in PERV-infected mink cells using electron microscopy together with numerous multivesicular body (MVB)-like structures containing small virus-like particles, not present in uninfected mink cells. These MVBs could be stained with PERV-specific serum. In an attempt to establish a small animal model, PERV grown on mink cells was inoculated into adult and newborn American minks. Neither antibody production against PERV nor integration of viral DNA or production of viral proteins in tissues of different organs could be detected 12 weeks post virus inoculation, indicating that PERV infection had not occurred.
Human-tropic porcine endogenous retroviruses (PERV) such as PERV-A and PERV-B can infect human cells and are therefore a potential risk to recipients of xenotransplants. A similar risk is posed by ...recombinant viruses containing the receptor-binding site of PERV-A and large parts of the genome of the ecotropic PERV-C including its long terminal repeat (LTR). We describe here the unique organization of the PERV-C LTR and its changes during serial passage of recombinant virus in human cells. An increase in virus titer correlated with an increase in LTR length, caused by multiplication of 37-bp repeats containing nuclear factor Y binding sites. Luciferase dual reporter assays revealed a correlation between the number of repeats and the extent of expression. No alterations have been observed in the receptor-binding site, indicating that the increased titer is due to the changes in the LTR. These data indicate that recombinant PERVs generated during infection of human cells can adapt and subsequently replicate with greater efficiency.
Porcine endogenous retroviruses (PERVs) are of particular concern with xenotransplantations using pig cells, tissues or organs as they are present in the genome of all pig strains and are able to ...infect human cells in vitro. However, it remains unclear whether PERV particles will be produced in vivo and whether they may infect xenotransplant recipients. Since normal pig peripheral blood mononuclear cells (PBMCs) may be transmitted together with the transplanted organ, the production of PERVs by stimulated PBMCs was studied in vitro.
To simulate antigen-induced activation of PBMCs, phytohaemagglutinin (PHA), a T cell mitogen, and the phorbol ester O-tetradecanoylphorbol-13-acetate (TPA), a tumour promoter, were used. Virus release was estimated by measuring reverse transcriptase (RT) activity and by RT-PCR of pelleted viruses.
Treatment of pig PBMCs with PHA or TPA induced the release of PERVs. For the first time, a correlation between the extent of proliferation of pig PBMCs and PERV production was shown. In addition, PERV release by non-proliferating cells and differences in virus production between stimuli as well as between different pig strains and individuals of one strain were observed. Subtype analysis revealed the release of the three subtypes PERV-A, PERV-B and PERV-C. In contrast to murine endogenous retroviruses, PERVs were induced by PHA alone.
The data suggest that the PBMCs transmitted within a xenotransplant may release PERV. These data also suggest that pig strains producing low amounts of virus could be more suitable for xenotransplantation.
: Xenotransplantation may be associated with the risk of transmission of microorganisms. In particular, the porcine endogenous retroviruses (PERV) have raised concerns as in vitro experiments show ...susceptibility of human cells for PERV infection. However, it remains unclear whether PERVs are able to infect transplant recipients in vivo and whether they are pathogenic. It is therefore essential that the risks are evaluated and for this purpose specific and sensitive screening methods for PERVs have to be developed. We generated specific antibodies against all major structural proteins of PERV and developed several assays which allow antibodies against PERV to be detected as indirect evidence of infection. For direct detection of PERV production, reverse transcriptase (RT) assays were used. PCR methods were used to detect provirus integration and the presence of viral mRNA. Here we present an immunoperoxidase assay (IPA), which would allow the detection of viral proteins in infected cells as well as antibodies against PERV in the serum of an infected host. The specificity of the sera used in the assay was determined by several methods, including immunoelectron microscopy, and the sensitivity of the assay was compared with other methods. This IPA was used to detect PERV infection in in vitro experiments for evaluation of the virus host range, for titrating the virus and for testing anti‐viral properties of AZT. Using this method it was shown that AZT inhibits replication of PERV. This IPA may be very useful for the surveillance of preclinical and clinical xenotransplantations.
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