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BACKGROUND: Decellularized human pulmonary heart valve (dhHV) scaffolds have been shown to be the gold standard especially for younger, adolescent patients. However, human heart valves are limited in availability. Xenogeneic decellularized pig heart valves (dpHV) may serve as alternative. METHODS: The efficacy of DNA reduction processes upon decellularization of heart valves from German Landrace pigs was analyzed by measurements of remaining nucleic acids including proviral porcine endogenous retrovirus (PERV) sequences. Porcine pulmonary heart valves (pPHV) were decellularized by three different protocols and further treated with DNaseI or Benzonase, at varying incubation times. DNA isolated from valve associated muscle (m), valve cusp (c), and pulmonary artery (pa) was monitored by PCR and qRT-PCR using GAPDH and the PERV polymerase (pol) for read-out. RESULTS: Decellularization of pPHV led to a significant reduction of DNA (>99%) which could be further significantly increased for (m) and (pa) by nuclease treatment, reducing proviral PERV pol from approximately 5 × 107 to 5 × 103 copies/mg in nuclease treated tissues. CONCLUSIONS: Both nucleases demonstrated comparable activities. But DNaseI revealed to be less consistent for PERV, especially at muscular tissue. Noteworthy, remaining proviral sequences are still detectable by PCR; however, due to the absence of the cellular replication machinery the production of infectious particles is not expected. Decellularization and nuclease treatment of pPHV is an efficient procedure to reduce the DNA content including PERV, thus represents a valuable option to increase virus safety independently from the source animal background.

Xenotransplantation of porcine cells, tissues, and organs may be associated with the transmission of porcine microorganisms to the human recipient. A previous, 2009, version of this consensus statement focused on strategies to prevent transmission of porcine endogenous retroviruses (PERVs). This version addresses potential transmission of all porcine microorganisms including monitoring of the recipient and provides suggested approaches to the monitoring and prevention of disease transmission. Prior analyses assumed that most microorganisms other than the endogenous retroviruses could be eliminated from donor animals under appropriate conditions which have been called "designated pathogen-free" (DPF) source animal production. PERVs integrated as proviruses in the genome of all pigs cannot be eliminated in that manner and represent a unique risk. Certain microorganisms are by nature difficult to eliminate even under DPF conditions; any such clinically relevant microorganisms should be included in pig screening programs. With the use of porcine islets in clinical trials, special consideration has to be given to the presence of microorganisms in the isolated islet tissue to be used and also to the potential use of encapsulation. It is proposed that microorganisms absent in the donor animals by sensitive microbiological examination do not need to be monitored in the transplant recipient; this will reduce costs and screening requirements. Valid detection assays for donor and manufacturing-derived microorganisms must be established. Special consideration is needed to preempt potential unknown pathogens which may pose a risk to the recipient. This statement summarizes the main achievements in the field since 2009 and focus on issues and solutions with microorganisms other than PERV.

Islet xenotransplantation represents an attractive solution to overcome the shortage of human islets for use in type 1 diabetes. The wide-scale application of clinical islet xenotransplantation, however, requires that such a procedure takes place in a specifically and tightly regulated environment. With a view to promoting the safe application of clinical islet xenotransplantation, a few years ago the International Xenotransplantation Association (IXA) published a Consensus Statement that outlined the key ethical and regulatory requirements to be satisfied before the initiation of xenotransplantation studies in diabetic patients. This earlier IXA Statement also documented a disparate regulatory landscape among different geographical areas. This situation clearly fell short of the 2004 World Health Assembly Resolution WHA57.18 that urged Member States "to cooperate in the formulation of recommendations and guidelines to harmonize global practices" to ensure the highest ethical and regulatory standards on a global scale. In this new IXA report, IXA members who are active in xenotransplantation research in their respective geographic areas herewith briefly describe changes in the regulatory frameworks that have taken place in the intervening period in the various geographic areas or countries. The key reassuring take-home message of the present report is that many countries have embraced the encouragement of the WHO to harmonize the procedures in a more global scale. Indeed, important regulatory changes have taken place or are in progress in several geographic areas that include Europe, Korea, Japan, and China. Such significant regulatory changes encompass the most diverse facets of the clinical application of xenotransplantation and comprise ethical aspects, source animals and product specifications, study supervision, sample archiving, patient follow-up and even insurance coverage in some legislations. All these measures are expected to provide a better care and protection of recipients of xenotransplants but also a higher safety profile to xenotransplantation procedures with an ultimate net gain in terms of international public health.

Background Porcine endogenous retroviruses (PERV) pose a zoonotic risk potential in pig-to-human xenotransplantation given that PERV capacity to infect different human cell lines in vitro has been clearly shown in the past. However, PERV infectious potential for human peripheral blood mononuclear cells (huPBMC) has been also demonstrated, albeit with controversial results. As productive PERV infection of huPBMC involves immune suppression that may attract opportunistic pathogens as shown for other retroviruses, it is crucial to ascertain unequivocally huPBMC susceptibility for PERV. To address this question, we first investigated in vitro infectivity of PERV for huPBMC using supernatants containing highly infectious PERV-A/C. Second, huPBMC were cocultivated with PERV-A/C producer cells to come a step closer to the in vivo situation of xenotransplantation. In addition, cocultivation of huPBMC with porcine PBMC (poPBMC) isolated from German landrace pigs was performed to distinguish PERV replication competence when they were constitutively produced by immortalized cells or by primary poPBMC. Methods Supernatants containing recombinant highly infectious PERV-A/C were used to infect PHA-activated huPBMC in the presence or absence of polybrene. Next, PERV-producing cell lines such as human 293/5° and primary mitogenically activated poPBMC of three German landrace pigs were cocultivated with huPBMC as well as with susceptible human and porcine cell lines as controls. PERV infection was monitored by using three test approaches. The presence of provirus DNA in putatively infected cells was detected via sensitive nested PCR. Viral expression was determined by screening for the activity of gammaretroviral reverse transcriptase (RT) in cell-free supernatants of infected cells. Virus release was monitored by counting the number of packaged RNA particles in supernatants via PERV-specific quantitative one-step real-time reverse transcriptase PCR. Results Porcine endogenous retroviruses-A/C in supernatants of human producer 293/5° cells was not able to infect huPBMC. Neither RT activity nor PERV copies were detected. Even provirus could not be detected displaying the inability of PERV-A/C to induce a productive infection in huPBMC. In cocultivation experiments only non-productive infection of huPBMC with PERV derived from 293/5° cell line and from PHA-activated poPBMC was observed by detection of provirus DNA in infected cells. Conclusion Recombinant PERV-A/C in supernatants of producer cells failed to infect huPBMC, whereas coculture experiments with producer cell lines lead to non-productive infection of huPBMC. PERV in supernatants seem to have not sufficient infectious potential for huPBMC. However, extensive PERV exposure to huPBMC via cocultivation enabled at least virus cell entry as provirus was detected by nested PCR. Furthermore, results presented support previous data showing German landrace pigs as low producers with negligible infectious potential due to the absence of replication-competent PERV in the genome. The low PERV expression profile and the lack of significant replication competence of German landrace pigs raise hope for considering these animals as putative donor animals in future pig-to-human xenotransplantation. Nonetheless, data imply that PERV still represent a virological risk in the course of xenotransplantation, as the presence of PERV provirus in host cells may lead to a provirus integration resulting in insertional mutagenesis and chromosomal rearrangements.

After feedback from the working parties, the final session focused on drafting proposed revisions of the WHO documents, and resulted in the formulation of the draft “Third WHO Global Consultation on Regulatory Requirements for Xenotransplantation Clinical Trials, The 2018 Changsha Communiqué.” This draft was submitted to WHO in February 2019 for WHO and World Health Assembly consideration. If approved, the 2018 Changsha Communiqué will then be posted on the websites of WHO, IXA, and TTS, and published in Xenotransplantation. This report includes summaries of the various sessions, followed by the abstracts of invited speakers from the update sessions.

Background For xenotransplantation, strategies to prevent transmission of microorganisms from the source animal to the human recipient must be closely coordinated since tissues and organs are classified as non-sterile. Strategies for international cooperation and coordination of xenogeneic infection / disease surveillance and response are available. Methods The regulatory frameworks and criteria on microbial safety as published by World Health Organization (WHO), European Pharmacopoeia (Ph. Eur.), European Medicines Agency (EMA) as well as U.S. Department of Health and Human Services (DHHS), Food and Drug Administration (FDA) and Center for Biologics Evaluation and Research (CBER), are outlined. Results Different sources of microbial germs are considered including potential infectious agents. Monitoring of livestock and testing of xenografts is accompanied by positive and negative controls to detect and to exclude tissue specific microorganisms such as bacteria. Conclusions The criteria of microbial status to be considered for xenotransplants are summarized.

Advances in xenotransplantation offer chances to alleviate the shortage of human donor organs. The discovery that pig endogenous retroviruses (PERV) can infect human cells in vitro has stimulated the discussion on infectious risk in xenotransplantation. A molecular and immunologic monitoring of xenograft recipients and of donor animals for putative infection with PERV and other microorganisms is inevitable. In this report, we describe the generation and testing of a highly specific anti-serum directed against the PERV nucleocapsid protein. The Gag amino acid (aa) sequence of PERV class B was used to define immunogenic domains by computer analysis. A peptide corresponding to the C-terminal 19 aa of the 10 kDa (p10) nucleocapsid (NC) portion of the Gag polyprotein was used to immunize rabbits. The generated serum was tested using recombinant PERV Gag protein expressed in insect cells, purified PERV virus particles and human 293 cells transfected or infected with PERV, respectively. Test methods included Western blotting, indirect immunofluorescence, immunoperoxidase assay and ELISA. The PERV anti-serum provides a tool that is instrumental for detection of a potential agent of zoonosis. It can be used for screening of donor animals and xenograft recipients in the course of xenotransplantation procedures.

Long terminal repeats (LTRs) of the human endogenous retrovirus K (HERV-K) family have been found at several sites within the human genome, of which one is located in the vicinity of HLA-DQB1. Since this DQLTR3 is only present on some haplotypes, we performed a linkage analysis in 130 Caucasian families. In order to date the integration event we also investigated the presence of this DQLTR3 in apes and Old World monkeys. Additionally, we sequenced the adjacent region of DQLTR3-positive and -negative haplotypes in humans, apes and old world monkeys to elucidate their evolution. Linkage analysis revealed a differential integration of DQLTR3 on specific HLA-DQ haploypes: there was a high frequency of this LTR on haplotypes containing HLA-DQB1*0302 (0.96) and a moderate frequency on HLA-DQB1*0402 (0.78), HLA-DQB1*0303 (0.44), HLA-DQB1*0502 (0.38) and HLA-DQB1*0301 (0.35). HLA-DQB1*0201 (0.18), HLA-DQB1*0503 (0.15), HLA-DQB1*0603 (0.15), HLA-DQB1*0602 (0.04), HLA-DQB1*0501 (0.03) and HLA-DQB1*0604 were rarely positive or devoid of DQLTR3. In apes and Old World primates there was no DQLTR3 rendering it a human specific insertion. Sequence analysis of the adjacent region showed two different motifs in humans corresponding to either presence or absence of DQLTR3. Two different motifs were observed within three sequences of Macaca mulatta: One motif is closely related to the sequence from Macaca nemestrina and Macaca fascicularis whereas the other sequence is more closely related with that of Papio papio and Cercopithecus aethiops. Therefore the analysis of retroviral elements as well as intronic sequences of MHC-DQB1 could help to clarify the evolution of this gene region as well the phylogenic relationship between humans, apes and Old World monkeys.

The linker histone H1degree accumulates in terminally differentiating cells and replaces other members of the H1 histone family, even in the absence of cell division. To study the role of H1degree in vivo, we have created two lines of transgenic mice with either the human H1degree promoter (HH minigene) or the mouse metallothionein T promoter (MH minigene) upstream of the human H1degree gene. Mice bearing the minigenes HH or MH overexpress human H1degree mRNA at 10-20-fold higher levels than in normal mice in a constitutive or metal-inducible manner. In contrast to this increase in mRNA content, which was studied in liver, kidney and brain, no significant changes in the relative proportions of the H1 protein subtypes, including H1degree were observed. Transgenic mice exhibited normal anatomic phenotypes, growth rates and reproduction rates. Thus, our results suggest a posttranscriptional and/or translational mechanism that compensates the unbalanced linker-histone expression in different tissues.

The transcription rates of the albumin and alpha-fetoprotein (alpha FP) genes were reduced to marginally detectable levels in livers of newborn or fetal c14CoS albino deletion mutant mice, which lack the hepatocyte specific developmental regulation (hsdr-1) locus on chromosome 7 and die shortly after birth. However, steady-state levels of these two mRNAs in livers of mutant mice were similar to those in normal mice, where these genes are actively transcribed. In c14CoS mice, transcription rates of transcription factor genes HNF-1, C/EBP and HNF-4 were reduced, albeit to different extents. These effects are specific because transcription of the HNF-3, DBP, LAP and Jun-B genes remained normal in mutant mice. Steady-state levels of all of these mRNAs reflected the transcriptional activities. Levels of HNF-1 and HNF-4 mRNAs showed much greater depression than that of C/EBP in mutant liver. The availability of this group of transcription factors may be reduced in c14CoS hepatocytes and therefore caused depressed transcription rates of their target genes such as those encoding albumin and alpha FP. However, the normal steady-state levels of albumin and alpha FP mRNAs in mutant mice remains unexplained. Fetal c14CoS hepatocytes in primary culture did acquire competence for glucocorticoid inducible transcription of the albumin, alpha FP, HNF-4 and metallothionein genes but not of the tyrosine aminotransferase (TAT) gene. These results indicate that the hsdr-1 locus is dispensable for the glucocorticoid induced transcription of these genes but not of TAT. The effects caused by the c14CoS deletion are pleiotropic in controlling the expression of numerous genes at distinct levels in the liver.

The life-cycles of viruses depend on factors provided by host cells due to their limited genetic coding capacity (1). The permissiveness of a host cell is determined by restriction factors that evolved during host-virus co-evolution (2). Several restriction factors have been shown to be important for the replication of retroviruses: TRIM5α disrupts the viral capsid (CA) after cell entry; TRIM28 blocks viral transcription; ZAP (zinc finger antiviral protein) directs degradation of viral RNAs; tetherin traps virions on the surfaces of infected cells; and APOBEC (apolipoprotein B mRNA-editing catalytic polypeptides) that are cytidine deaminases disrupts viral DNA during synthesis (2, 3). PERV-A and recombinant PERV-A/C are insensitive to restriction by TRIM5α in permissive feline cells expressing TRIMα proteins from humans, Africa green monkeys, rhesus macaques, squirrel monkeys, rabbits, or cattle (4). On the other hand, overexpression of either human or porcine tetherin in pig cells significantly reduced PERV production (5). The mammalian APOBEC3 (A3) genes are part of the AID/APOBEC gene family. Their members share structural and functional domains of zinc-dependent deaminases (6). Proteins of the A3 group contain one or two zinc (Z)-coordinating domains and are classified according to the presence/absence of a Z1, Z2, or Z3 motif (7, 8). Initial studies showed that porcine poA3Z2-Z3 did not significantly interfere with PERV transmission (9) and it was concluded that PERV was resistant to its species-specific A3 protein (10). Subsequently, the chromosomal porcine A3 locus for poA3Z2 and poA3Z3 was reanalyzed and data showed that pigs express four different A3 mRNAs, encoding poA3Z2 and poA3Z3 and, by readthrough transcription and alternative splicing, poA3Z2-Z3 and poA3Z2-Z3 splice variant A (SVA) (11, 12). Results illustrated that PERV was significantly inhibited by various porcine A3s in single-round as well as spreading virus assays. PERV inhibition strongly correlated with a specific cytidine deamination in viral genomes for the trinucleotides 5′TGC, for poA3Z2 as well as poA3Z2-Z3, and 5′CAC, for A3Z3 (11, 12). Data demonstrate that human and porcine A3s can inhibit PERV replication in vivo, thereby reducing the risk of potential infection of human cells by PERV in the course of pig-to-human xenotransplantation. References: 1. Watanabe T, Watanabe S, Kawaoka Y. Cellular networks involved in the influenza virus life cycle. Cell Host Microbe 2010; 7: 427–439. 2. Wolf D, Goff SP. Host restriction factors blocking retroviral replication. Annu. Rev. Genet. 2008; 42: 143–163. 3. Meije Y, TOnjes RR, Fishman JA. Retroviral restriction factors and infectious risk in xenotransplantation. Am. J. Transplant. 2010; 10: 1511–1516. 4. Wood A, Webb BLJ, Bartosch B, Schaller T, Takeuchi Y, Towers GJ. Porcine endogenous retroviruses PERV A and A/C recombinant are insensitive to a range of divergent mammalian TRIM5a proteins including human TRIM5a. J. Gen. Virol. 2009; 90: 702–709. 5. Mattiuzzo G, Ivol S, Takeuchi Y. Regulation of porcine endogenous retrovirus release by porcine and human tetherins. J. Virol. 2010; 84: 2618–2622. 6. Conticello SG, Thomas CJ, Petersen-Mahrt SK, Neuberger MS. Evolution of the AID/APOBEC family of polynucleotide (deoxy)cytidine deaminases. Mol. Biol. Evol. 2005; 22: 367–377. 7. LARUE RS, ANDRESDOTTIR V, BLANCHARD Y, CONTICELLO SG, DERSE D, EMERMAN M, GREENE WC, JONSSON SR, LANDAU NR, LOCHELT M, MALIK HS, MALIM MH, MUNK C, O’BRIEN SJ, PATHAK VK, STREBEL K, WAIN-HOBSON S, YU XF, YUHKI N, HARRIS RS. Guidelines for naming nonprimate APOBEC3 genes and proteins. J. Virol. 2009; 83: 494–497. 8. MUnk C, Beck T, Zielonka J, Hotz-Wagenblatt A, Chareza S, Battenberg M, Thielebein J, Cichutek K, Bravo IG, O’Brien SJ, LOchelt M, Yuhki N. Functions, structure, and read-through alternative splicing of feline APOBEC3 genes. Genome Biol. 2008; 9: R48. 9. JOnsson SR, HachEG, Stenglein MD, Fahrenkrug SC, AndrEsdOttir V, Harris RS. Evolutionarily conserved and non-conserved retrovirus restriction activities of artiodactyl APOBEC3F proteins. Nucleic Acids Res. 2006; 34: 5683–5694. 10. JOnsson SR, LaRue RS, Stenglein MD, Fahrenkrug SC, AndrEsdOttir V, Harris RS. The restriction of zoonotic PERV transmission by human APOBEC3G. PLoS One 2007; 2: e893. 11. DOrrschuck E, MUnk C, TOnjes RR. APOBEC3 proteins and porcine endogenous retroviruses. Transplant. Proc. 2008; 40: 959 –961. 12. DOrrschuck E, Fischer N, Bravo IG, Hanschmann KM, Kuiper H, SpOtter A, MOller R, Cichutek K, MUnk C, TOnjes RR. Restriction of porcine endogenous retrovirus by porcine APOBEC3 cytidine deaminases. J. Virol. 2011; 85: 3842–3857.

Porcine endogenous retroviruses (PERV) pose a xenozoonotic risk when applying pig organs, tissues and cells in clinical xenotransplantation. Three classes of replication-competent PERV were described in host range and interference studies [1]. Polytropic PERV-A and PERV-B are able to infect not only human cells but also various cell lines in vitro using host membrane proteins as receptors [2]. Although cell tropism of ecotropic PERV-C is mainly restricted to porcine cells, PERV-C represents a considerable infectious risk. On the one hand, PERV-C serves as template for recombination with PERV-A resulting in highly infectious human-tropic PERV-A/C [3]. On the other hand, PERV-C may evolve towards an infectious human-tropic variant since the PERV-C receptor-binding domain could be bound to human cells and solely four amino acid exchanges in the surface unit of the envC gene are sufficient to permit receptor-mediated membrane fusion and virus entry [4]. To guarantee retroviral safety in pig-to-human xenotransplantation generation of appropriate pigs free from replication-competent PERV-A and PERV-B as well as ecotropic PERV-C is required. Using PCR-based methods and directional cloning strategies, we cloned and characterized PERV-C. Genomic DNA was prepared from peripheral blood mononuclear cells derived from three different pig subspecies which are either already used or intended for clinical xenotransplantation. One PERV-C clone was reconstructed using genomic DNA of an Auckland Islands pig derived from a DPF herd in New Zealand. Islet cell clusters of these PERV non-transmitters are used in clinical trials to treat diabetes type I patients. Moreover, the potential of brain cells from these animals to treat Parkinson's and Huntington's disease is currently tested clinically. Similarly, a PERV-C clone was reconstructed from genomic DNA of Gottingen minipig which is a PERV non-transmitter as tested in co-cultures with susceptible human HEK 293 cells [5]. Finally, a bacteriophage lambda library was constructed from genomic DNA of a d/d haplotype miniature pig. The individuals of this pig line show lower PERV transmission levels in vitro [6]. We isolated a λ-clone containing PERV-C 5′-LTR, gag, pro/pol as well as large part of the envC gene compared to replication-competent PERV-C(1312) [7]. The provirus is truncated due to our cloning strategy. Nonetheless, using PCR and PERV-C specific primers the missing part of the envC gene and 3′-LTR was amplified and ligated to the proviral fragment present in the λ-clone. Replication-competence of reconstructed full-length viruses is currently tested in susceptible ST-Iowa cells. References [1] Takeuchi Y, Patience C, Magre S et al. Host range and interference studies of three classes of pig endogenous retrovirus. J Virol. 1998; 72: 9986–9991. [2] Ericsson TA, Takeuchi Y, Templin C et al. Identification of receptors for pig endogenous retrovirus. Proc Natl Acad Sci U S A 2003; 100: 6759–6764. [3] Wilson CA, Wong S, Vanbrocklin M, Federspiel MJ. Extended analysis of the in vitro tropism of porcine endogenous retrovirus. J Virol. 2000; 74: 49–56. [4] Argaw T, Figueroa M, Salomon DA, Wilson CA. Identification of residues outside of the receptor binding domain that influence the infectivity and tropism of porcine endogenous retroviruses. J Virol. 2008; 82: 7483–7491. [5] Semaan M, Rotem A, Barkai U, Bornstein S, Denner J. Screening pigs for xenotransplantation: prevalence and expression of porcine endogenous retroviruses in Gottingen minipigs. Xenotransplantation. 2013; 20: 148–156. [6] Oldmixon BA, Wood JC, Ericsson TA et al. Porcine endogenous retrovirus transmission characteristics of an inbred herd of miniature swine. J Virol. 2002; 76: 3045–3048. [7] Preuss T, Fischer N, Boller K, Tonjes RR. Isolation and characterization of an infectious replication-competent molecular clone of ecotropic porcine endogenous retrovirus class C. J Virol. 2006; 80: 10258–10261.

Pig to human xenotransplantation represents an ambitious venture that requires, besides evasion of rejection mechanisms and physiological incompatibilities, the generation of pathogen-free pigs as donors for well characterized xenografts to warrant medicinal products that do comply with statutory regulatory demands [1–3]. The publication of a high quality draft sequence for the pig genome (Sus scrofa) and a series of accompanying reports for the first time offered the feasibility of whole genome expression profiling of porcine tissues and cells [4–8]. The SFB TR CRC 127 project Z2 “Microbiological Safety including Virological Safety” is based on microbial profile analysis of porcine tissues in order to prevent zoonotic events, including infection by porcine endogenous retroviruses (PERV) [9]. The project comprises the detection and characterization of potential pathogens as well as the investigation of the microbial influence on the transcriptional status of tissues and cells. Hence, specific expression patterns, e.g. up-regulation of antiviral host factors or cell cycle/apoptotic regulators, may also provide information on ongoing or precedent events that may have impact on tissues/cells quality and therefore its suitability as xenografts. We use microarray technology for monitoring viability of tissues and cells as well as their microbial/viral status. An Agilent based, 60K DNA microarray representing 25,415 different genes of the recently published Sus scrofa genome (NCBI Sus scrofa 10.2-assembly) was generated [10]. The microarray was specified for German Landrace and Gottingen Minipig, amongst other pig species, by hybridizing complex RNA samples generated from five different pig organs and blood as well as chromosomal porcine DNA to highlight non expressed genes. Four Diagnostic PERV sequences for pro/pol (all classes of PERV), env (to differentiate between PERV-A, -B and -C) as well as 15 human transgenes such as CD59 (human complement regulatory protein), DAF (Decay accelerating factor or CD55), human A20 (hA20) and others were included. In total, the microarray displays 25,434 genes each represented by up to three different 60-mer oligonucleotides. To reveal functionality of the microarray the transcriptional status of ST-IOWA cells freshly infected with molecularly cloned virus PERV-C (1312) [11] was monitored. Total mRNA levels at day 7, 28 and 56 post infection were compared with naive uninfected cells. All samples were tested in triplicates and the relative signal intensity of hybridized probes was compared. Special attention was given to antiviral host factors such as APOBEC and tetherin of which involvement as antiviral factors on PERV expression has been demonstrated [12–14]. Constitutively expressed housekeeping genes, i.e. porcine glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta actin and cyclophilin A, respectively, were used as controls [15, 16]. The presented microarray supports the safety and quality by monitoring the transcriptional status of xenotransplants. References [1] EMEA/CHMP/CPWP/83508/2009. Guideline on Xenogeneic Cell-Based Medicinal Products. [2] U.S. Department of Health and Human Services. Food and Drug Administration. Center for Biologics Evaluation and Research (CBER). February 2002. Draft Guidance for Industry: Precautionary Measures to Reduce the Possible Risk of Transmission of Zoonoses by Blood and Blood Products from Xenotransplantation Product Recipients and Their Intimate Contacts. [3] U.S. Department of Health and Human Services. Food and Drug Administration. Center for Biologics Evaluation and Research (CBER). April 2003. Guidance for Industry: Source Animal, Product, Preclinical, and Clinical Issues Concerning the Use of Xenotransplantation Products in Humans. [4] Groenen MA, et al. Analyses of pig genomes provide insight into porcine demography and evolution. Nature 2012; 491(7424): 393–398. [5] Li Y, Mei S, Zhang X, et al. Identification of genome-wide copy number variations among diverse pig breeds by array CGH.BMC Genomics 2012; 13:725. doi: 10.1186/1471-2164-13-725. [6] Servin B, Faraut T, Iannuccelli N, Zelenika D, Milan D. High-resolution autosomal radiation hybrid maps of the pig genome and their contribution to the genome sequence assembly. BMC Genomics 2012; 13:585. doi: 10.1186/1471-2164-13-585. [7] Nguyen DT, Lee K, Choi H, et al. The complete swine olfactory subgenome: expansion of the olfactory gene repertoire in the pig genome. BMC Genomics 2012; 13:584. doi: 10.1186/1471-2164-13-584. [8] Uenishi H, Morozumi T, Toki D et al. Large-scale sequencing based on full-length-enriched cDNA libraries in pigs: contribution to annotation of the pig genome draft sequence. BMC Genomics 2012; 13:581. doi: 10.1186/1471-2164-13-581. [9] Denner J, Tonjes RR. Infection barriers to successful xenotransplantation focusing on porcine endogenous retroviruses. Clin Microbiol Rev. 2012; 25(2): 318–743. [10] Agilent's Microarray Platform. How High-Fidelity DNA Synthesis Maximizes the Dynamic Range of Gene Expression Measurements. Library – Application Note. 2013; Publication Part Number: 5989-9159EN [11] Preuss T, Fischer N, Boller K, Tonjes RR. Isolation and characterization of an infectious replication-competent molecular clone of ecotropic porcine endogenous retrovirus class C. J Virol. 2006; 80(20):10258–61. [12] Dorrschuck E, Fischer N, Bravo IG, et al. Restriction of porcine endogenous retrovirus by porcine APOBEC3 cytidine deaminases. J Virol. 2011; 85(8):3842–57. [13] Dorrschuck E, Munk C, Tonjes RR. APOBEC3 proteins and porcine endogenous retroviruses. Transplant Proc. 2008; 40(4):959–61. [14] Mattiuzzo G, Ivol S, Takeuchi Y. Regulation of porcine endogenous retrovirus release by porcine and human tetherins. J. Virol. 2010; 84: 2618 –2622. [15] Mcculloch RS, S. Ashwell M, O'Nan AT, Mente PL. Identification of stable normalization genes for quantitative real-time PCR in porcine articular cartilage. J Anim Sci Biotechnol. 2012; 3(1):36. [16] Nygard AB, Jorgensen CB, Cirera S, Fredholm M. Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR. BMC Mol Biol. 2007; 8:67.

The clinical application of xenotransplantation evokes immunological and microbiological as well as virological challenges. Porcine pathogens that do not show any symptoms in their natural host could exhibit a risk of fatal infections to humans. The presence of pig infectious agents including zoonotic and dissimilar agents should be reduced by specific pathogen free (spf) breeding of donor animals. However, the genetic information of porcine endogenous retroviruses (PERV) is integrated in the pig genome and can not be eradicated by spf breeding. The concerns about PERV for human xenograft recipients are based on data of in vitro replication of PERV in some human cell lines. So far, viral replication of PERV has been difficult to demonstrate in non-human primate cell lines and in preclinical studies of non-human primates receiving porcine xenografts, respectively. In this regard, natural and effective mechanisms of human and porcine cells counteracting productive infections caused by PERV are important to investigate. Intracellular proteins and components of the innate immune system including endogenous “antiretroviral restriction factors” act at various steps in retroviral replication. The cellular front is composed by several constitutively expressed genes which prevent or suppress retroviral infections. Some of these factors such as members of the tripartite motif (TRIM) and the apolipoprotein B mRNA-editing polypeptide (APOBEC) families as well as tetherin and zinc-finger antiviral protein (ZAP) could be useful in the management of PERV in xenotransplantation. The risks of infection and the potential role of antiretroviral restriction factors in xenotransplantation are presented in detail.

Human cells might display mechanisms counteracting infections by porcine endogenous retroviruses (PERV) in the course of pig-to-human xenotransplantation. Mammals have developed a number of protective strategies against viruses, including an intracellular antiretroviral defense as part of the innate immunity. In addition to the conventional innate and acquired immune responses an array of dominant genes have evolved that are constitutively expressed which suppress or prevent retroviral infections. Several of these antiretroviral restriction mechanisms have been identified including members of the tripartite motif (TRIM) and APOBEC families. The TRIM5 class of inhibitors appears to target incoming retroviral capsids and the APOBEC class of cytidine deaminases hypermutates and destabilizes retroviral genomes. Our data show that human and porcine cytidine deaminases inhibit PERV replication significantly, thereby reducing the infectious risk raised by PERV in vitro. The exact mechanism of the TRIM5 mediated restriction has not been exactly determined so far. Data published by Wood et al. (2009) indicate that PERV are insensitive to restriction by divergent TRIM5 molecules including human and monkey TRIM5α?proteins. The role of pig TRIM5 has not been clarified. We have identified a single TRIM5 gene in the pig genome. The impact of porcine TRIM5 protein on PERV will be tested using the human TRIM5α as a negative control, expecting that PERV will be insensitive to porcine TRIM5.

Zoonoses pose a threat to mammalian species. Cross-species transmission of viruses have given rise to fatal diseases because the host organism is not prepared to resist a new pathogen. Mammals have developed several strategies of defense against viruses, including an intracellular antiretroviral defense, a part of innate immunity. In addition to the conventional innate and acquired immune responses, complex organisms such as mice and primates have evolved an array of dominant, constitutively expressed genes that suppress or prevent retroviral infections. Several of these antiretroviral restriction mechanisms have recently been identified, with two particularly well described factors being members of the tripartite motif (TRIM) and APOBEC families. The TRIM5 class of inhibitors appears to target incoming retroviral capsids and the APOBEC class of cytidine deaminases hypermutates and destabilizes retroviral genomes. Lentiviruses such as HIV-1 have developed countermeasures that allow them to replicate despite the human host factors. In the course of risk assessment for pig-to-human xenotransplantation the capacity of human cells to counteract infections of gamma-type porcine endogenous retroviruses (PERV) should be analyzed. We raised the question as to whether PERV is affected by APOBEC3 proteins. Initial data indicate that human and porcine cytidine deaminases inhibit PERV replication, thereby possibly reducing the risk for infection of human cells by PERV as a consequence of pig-to-human xenotransplantation. The exact mechanism of the TRIM5 mediated restriction has not been clarified up to now. At current, we investigate how many TRIM5 genes are located in the pig genome. Furthermore, the properties of porcine TRIM5α isoform proteins will be tested and we will check the potential of the human TRIM5α to restrict PERVs in order to determine the risk of virus transmission.

Deletions in chromosome 7 of the mouse have been shown to cause failure of expression of various hepatocyte-specific genes in newborn deletion homozygotes, including the gene encoding tyrosine amino transferase (TAT) (EC 2.6.1.5) (Gluecksohn-Waelsch, 1979). Primary liver cultures of newborn albino deletion mutant mice (c14CoS/c14CoS) and of phenotypically normal mice (c14CoS/cch or cch/cch) were infected with SV40 virus and multiplying hepatocytes selected in arginine-deficient medium containing epidermal growth factor (EGF), insulin, and hydrocortisone (HC). Resulting normal (NMH-ch) and mutant (NMH-m14) hepatocyte lines expressing integrated viral transforming sequences did not senesce, they multiplied autonomously of EGF in medium with insulin plus HC, and they retained hepatocyte-specific functions. Both lines synthesized arginine and contained albumin and alpha-fetoprotein (AFP) mRNAs. TAT-specific mRNA was detected in normal but not in mutant hepatocyte lines. A fragment of the mouse tyrosinase gene, known to map at the albino locus (c) within the region deleted in the c14CoS mutant, hybridized with a 2.5 kb EcoRI fragment of normal NMH-ch DNA, whereas this fragment was undetectable in mutant NMH-m14 DNA. These immortalized hepatocyte lines reflect important properties of normal and mutant liver tissues from which they were derived. The deletion mutant mouse cell lines may be useful for complementation studies involving sequences corresponding to the deletions that encode regulatory gene(s) involved in the control of inducible expression of certain hepatocyte-specific genes such as TAT.