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INTRODUCTION: Porcine endogenous retroviruses (PERVs) are an integral part of the pig genome with infectious potential, as shown in vitro. HYPOTHESIS/GAP STATEMENT: In view of nonclinical and clinical xenotransplantation, data are essential that give an insight into viral pathogenicity. This includes PERV's environmental stability and environmental risk.AIM: We analyzed two ecotropic PERV-C (PERV-C[1312] and -[5683]), monitoring cell-free culture supernatants of infected ST-IOWA cells at various time intervals at room temperature (22°C +/−1°C). The virus was stored in the presence or absence of sterile wood litter, as used for large animal husbandry. This approach was set to determine the environmental stability of exogenous PERV-C at defined conditions for the first time. METHODOLOGY: Reverse transcriptase (RT) activity and viral RNA were monitored for up to 57 days and remaining infectivity of supernatant without wood litter was tested from day 7 onwards on naïve ST-IOWA cells. RESULTS: Results show that viral RNA decreases but remains detectable over the whole observation period, whereas RT activity showed 83%–96% reduction from day 7 on. This effect was stronger in the presence of wood litter and fresh harvested virus was more stable than frozen virus stocks. Even under these optimal conditions, no infectivity was shown for viral RNA-positive and RT-reduced supernatant harvested at day 7. CONCLUSION: The results confirm that PERV-C is less stable and the reduction of RT activity is accompanied by reduced infectivity, independently of existing viral RNA. The combination of both RT and viral RNA measurement is a suitable method to differentiate infectious PERV-C.

Background The demand for donated human hearts far exceeds the number available. Xenotransplantation of genetically modified porcine organs provides an alternative. In 2000, an Advisory Board of the International Society for Heart and Lung Transplantation set the benchmark for commencing clinical cardiac xenotransplantation as consistent 60% survival of non-human primates after life-supporting porcine heart transplantations. Recently, we reported the stepwise optimization of pig-to-baboon orthotopic cardiac xenotransplantation finally resulting in consistent success, with 4 recipients surviving 90 (n = 2), 182, and 195 days. Here, we report on 4 additional recipients, supporting the efficacy of our procedure. Results The first 2 additional recipients succumbed to porcine cytomegalovirus (PCMV) infections on Days 15 and 27, respectively. In 2 further experiments, PCMV infections were successfully avoided, and 3-months survival was achieved. Throughout all the long-term experiments, heart, liver, and renal functions remained within normal ranges. Post-mortem cardiac diameters were slightly increased when compared with that at the time of transplantation but with no detrimental effect. There were no signs of thrombotic microangiopathy. The current regimen enabled the prolonged survival and function of orthotopic cardiac xenografts in altogether 6 of 8 baboons, of which 4 were now added. These results exceed the threshold set by the Advisory Board of the International Society for Heart and Lung Transplantation. Conclusions The results of our current and previous experimental cardiac xenotransplantations together fulfill for the first time the pre-clinical efficacy suggestions. PCMV-positive donor animals must be avoided.

Xenotransplantation of porcine organs, tissues, and cells inherits a risk for xenozoonotic infections. Viable tissues and cells intended for transplantation have to be considered as potentially contaminated non-sterile products. The demands on microbial testing, based on the regulatory requirements, are often challenging due to a restricted shelf life or the complexity of the product itself. In Europe, the regulatory framework for xenogeneic cell therapy is based on the advanced therapy medicinal products (ATMP) regulation (2007), the EMA CHMP Guideline on xenogeneic cell-based medicinal products (2009), as well as the WHO and Council of Europe recommendations. In the USA, FDA guidance for industry (2003) regulates the use of xenotransplants. To comply with the regulations, validated test methods need to be established that reveal the microbial status of a transplant within its given shelf life, complemented by strictly defined action alert limits and supported by breeding in specific pathogen-free (SPF) facilities. In this review, we focus on assays for the detection of the porcine endogenous retroviruses PERV-A/-B/-C, which exhibit highly polymorphic proviral loci in pig genomes. PERVs are transmitted vertically and cannot be completely eliminated by breeding or gene knock out technology. PERVs entail a public health concern that will persist even if no evidence of PERV infection of xenotransplant recipients in vivo has been revealed yet. Nevertheless, infectious risks must be minimized by full assessment of pigs as donors by combining different molecular screening assays for sensitive and specific detection as well as a functional analysis of the infectivity of PERV including an adequate monitoring of recipients.

SUMMARY Xenotransplantation may be a solution to overcome the shortage of organs for the treatment of patients with organ failure, but it may be associated with the transmission of porcine microorganisms and the development of xenozoonoses. Whereas most microorganisms may be eliminated by pathogen-free breeding of the donor animals, porcine endogenous retroviruses (PERVs) cannot be eliminated, since these are integrated into the genomes of all pigs. Human-tropic PERV-A and -B are present in all pigs and are able to infect human cells. Infection of ecotropic PERV-C is limited to pig cells. PERVs may adapt to host cells by varying the number of LTR-binding transcription factor binding sites. Like all retroviruses, they may induce tumors and/or immunodeficiencies. To date, all experimental, preclinical, and clinical xenotransplantations using pig cells, tissues, and organs have not shown transmission of PERV. Highly sensitive and specific methods have been developed to analyze the PERV status of donor pigs and to monitor recipients for PERV infection. Strategies have been developed to prevent PERV transmission, including selection of PERV-C-negative, low-producer pigs, generation of an effective vaccine, selection of effective antiretrovirals, and generation of animals transgenic for a PERV-specific short hairpin RNA inhibiting PERV expression by RNA interference.

Xenotransplantation involves the transfer of cells, tissues, or organs from one species to another for therapeutic reasons. While the technology is under development, it has the potential to overcome the significant shortage of human allotransplants (31). Using the pig (Sus scrofa; order, Suidae) as the favored donor animal presents a number of important microbiological safety issues (40) which may be solved regarding exogenous agents by proper handling of the animals, such as barrier breeding according to specific-pathogen-free conditions and application of vaccinations (38). However, porcine endogenous retroviruses (PERV) pose a more serious challenge, since they are present in almost every individual (26) and are transmitted vertically. Endogenous retroviruses (ERV) are present in the genomes of all vertebrate species analyzed (8), and their general organization corresponds to exogenous retroviruses (8, 35, 41). Whereby multiple copies of ERV are present in their host genomes, most are truncated or mutated, rendering them replication incompetent. Nevertheless, some of the viral integration sites still display transcriptional activity and even produce viral particles by complementation in trans. Only a minority of the distinct proviruses are functional as reported for pigs (1, 2, 20, 25, 28, 29). While PERV belong to the gamma-retroviruses, other ERV also share homologies with beta- and delta-retroviruses but not with lentiviruses (12, 16, 28, 41). For PERV, three different classes, designated PERV-A, PERV-B, and PERV-C (1, 22), exist, whereby PERV-A and -B are polytropic and productively infect human cells in vitro, thus posing a serious risk in xenotransplantation and xenogeneic cell therapies. Ecotropic PERV-C (1) does not replicate on human cells and is therefore not included in this study. There are only minor genetic differences between the classes, which are most prominent in the receptor binding domain of the env protein (Fig. 1C and D). In addition, there are two different types of long terminal repeats (LTR) (Fig. ​(Fig.1B)1B) that significantly affect the replication properties of single viruses (33) by instrumentalizing a special set of transcription factors (32). Both PERV-A and PERV-B proviruses demonstrate LTRs that harbor repeats in U3. On the other hand, PERV-A and PERV-C were found to display repeatless LTRs (33). LTRs displaying repeat structures and possible multimerization mechanisms were first described for murine retroviruses (23, 43). These retroviruses, along with other gamma-type retroviruses, share a common homology of approximately 60% among each other and with PERV. Furthermore, so as not to complicate the analysis, we excluded any of these viruses from this study and concentrated on determining the relative age of the two different PERV LTR types not found in any other virus. It was shown that PERV in cell culture actively adapt their LTR repeat structure, if present, to match the optimal but not the maximum replication performance in a given host cell (33). Conversely, the recombinant incorporation of an artificially created repeat structure, i.e., 10 times 39 bp, into U3 of the 5′ LTR of a molecular clone caused rapid cell death after transfection into susceptible cells (Scheef and Tonjes, unpublished data). While different env isoforms are present in a variety of retroviruses (7), only PERV is known to harbor two related but profoundly different LTR types in addition to three env classes. FIG. 1. Genomic organization of proviral PERV. (A) PERV displays genes for group specific antigen (gag), protease/polymerase polyprotein (pro/pol), and envelope protein (env), flanked by LTR. Both LTR and env vary significantly between individual proviruses, ... In anthropological studies, human endogenous retroviruses (HERV) have been used successfully to designate the age of the proviruses in hominoids as well as the time points of species separation events and phylogenetic relationships of different hominoids (17, 18, 34). These studies had the benefit of comparing many different HERV from different hominoids, ranging from humans to apes to monkeys. To repeat such a study for PERV is complicated due to the present diversity of the Artiodactyla (27, 37). Therefore, the closest relatives to pigs, the American-born peccaries (Tayassuidae, Pecari tajacu; see below), were included in this study to analyze an archaeological fixed relationship between two species. Besides determining the age of PERV, we were particularly interested in investigating which of the two LTR types is the phylogenetic predecessor. We have analyzed the prevalence of six well-characterized full-length PERV, five of them being replication competent and four of them being chromosomally assigned (20, 25). These analyses revealed a heterogeneous distribution of PERV among individuals (26), and since no PERV is present in every pig, it seems feasible to generate pigs free of functional PERV by conventional breeding. In addition, specific proviruses show internal point mutations which significantly affect their replication capacities. Since there are two different types of PERV LTR structures (Fig. ​(Fig.1B)1B) showing various levels of transcriptional capacity (33), an analysis of 21 distinct chromosomal locations revealed that PERV which harbor highly active LTR with repeat elements in U3 are dominant (26). In addition, the two polytropic envelope genes were assayed for sequence variations, displaying class-specific hot spots of variation, as well as variations in the R peptide region (Fig. 1C and D).

Recent studies revealed a significant promoter activity of porcine endogenous retrovirus (PERV) long terminal repeats (LTRs) in different human and mammalian cell lines, which is mediated by a 39-bp repeat located in the U3 region in different numbers, representing an enhancer (G. Scheef, N. Fischer, U. Krach, and R. R. Tönjes, J. Virol. 75:6933-6940, 2001). A statistical transcription factor analysis revealed putative binding sites for the CCAAT-binding transcription factor NF-Y inside the 39-bp repeat. Specific binding of NF-Y to the repeat sequence was demonstrated by electrophoretic mobility shift assays and supershift assays with specific antibodies directed against the three subunits of NF-Y. To identify further transcription-regulating elements, genetically modified LTRs lacking the repeat box, U3, R, or U5 were investigated. The results indicated a strong inhibitory element in the R region, as the deletion of R caused a significantly increased promoter activity. Since PERV might play a potential role in the application of xenogeneic cell therapy and xenotransplantation techniques, we have investigated whether immunosuppressive drugs that are routinely used in transplantation medicine have an impact on the promoter activity. Neither cyclosporine nor prednisolone had any influence on the promoter strength of the PERV LTRs. By performing a real-time PCR we were able to compare the proviral loads of porcine and infected human cells as well as the amount of released virions, which revealed a direct link between LTR activity and the number of released retroviruses.

The organization and transcriptional regulation of porcine endogenous retrovirus (PERV) long terminal repeats (LTRs) are unknown. We have studied the activity of LTRs from replication-competent molecular clones by performing luciferase reporter assays. The LTRs differ in the presence and number of 39-bp repeats located in U3 that confer strong promoter activity in human, simian, canine, feline, and porcine cell lines, whereas for LTRs devoid of the repeats, the promoter strength was significantly reduced. As the activity of a heterologous simian virus 40 promoter and a homologous repeat-deficient LTR was elevated by four 39-bp repeats independently of its orientation and location, the repeat box complies with the definition of an enhancer. During serial virus passaging of molecular PERV clones on human 293 cells, proviral LTRs demonstrated adaptation of transcriptional activity by dynamic changes of the number of 39-bp repeats in the course of up to 12 passaging cycles.

We have established spontaneously formed B lymphoblastoid cell lines from long-term cultured peripheral blood mononuclear cells (PBMNCs) from multiple sclerosis (MS) patients. The MS cell lines actively produce retrovirus-like particles as well as Epstein-Barr virus (EBV). Using three different variations of the highly sensitive polymerase chain reaction (PCR)-based assays for the detection of reverse transcriptase (RT) activity, we have verified the retroviral origin of the retrovirus-like particles that are produced in very low amounts by the MS cell lines.

Differentiated human teratocarcinoma cell lines produce the human teratocarcinoma-derived virus (HTDV) particles encoded by the human endogenous retrovirus sequence HERV-K. We screened almost 2,000 human sera for antibodies against this endogenous human retrovirus, HTDV/HERV-K. Specificity of the immunofluorescence reactions using particle producing teratocarcinoma cells was confirmed by immunoelectron microscopy of ultrathin frozen sections. Immunoblot analyses using lysates of HTDV-producing cells revealed a 80-kDa HERV-K Gag precursor and a 90-kDa putative viral Env protein after incubation with positive sera. No processed Gag protein could be observed. Virus-specific bands were not detected in lysates of nonproducing cells. High antibody titers were found in about 60% of male patients with germ cell tumors. Antibody reactivity declined after tumor removal. In healthy blood donors, anti-HTDV reactivity was found only at low titers in a small percentage (3.9%) of individuals. A slightly elevated but statistically significant percentage of HTDV positivity was also observed for sera of pregnant women, whereas human immunodeficiency virus-positive individuals exhibited no peculiarity compared to normal blood donors. Our results provide evidence that HTDV particles are expressed in vivo and that the immune reaction against HTDV/HERV-K is specific for defined viral proteins.

The human endogenous retrovirus family HTDV/HERV-K codes for the viral particles observed in teratocarcinoma cell lines. Two types of proviral genomes exist; these differ in the presence or absence of a stretch of 292 nucleotides. This sequence comprises the amino-terminal part of the env gene, the putative signal peptide, which overlaps in part with the carboxy terminus of the pol gene. Type 2 genomes containing this sequence presumably more closely reflect the structure of the infectious, replication-competent retrovirus ancestors of the HERV-K family than do type 1 genomes that lack the sequence. In human teratocarcinoma cell lines, both variants are expressed. Type 1 genomes, in which pol and env genes are fused, are deficient in splicing. Type 2 transcripts are spliced to subgenomic env mRNA and smaller messages. A doubly spliced transcript encodes a short open reading frame, preliminarily designated cORF (R. Löwer, K. Boller, B. Hasenmeier, C. Korbmacher, N. Mueller-Lantzsch, J. Löwer, and R. Kurth, Proc. Natl. Acad. Sci. USA 90:4480-4484). The genomic organization of cORF resembles that of nonprimate lentivirus rev genes: the first exon comprises nearly the entire signal peptide of env, and the second exon is derived from a different reading frame in the 3' part of the genome. A nucleolar localization signal, which is also a putative RNA binding domain, as well as a sequence with similarities to the Rev effector domain consensus sequence is present in the first exon. Secondary structure analysis reveals similarities to basic helix-loop-helix proteins. cORF is a small protein with an apparent molecular mass of 14 kDa which accumulates in the nucleolus as has been described for Rev proteins.

The family of human endogenous retrovirus type K (HERV-K) comprises members with long open reading frames (ORF) for retroviral proteins. The existence of a biologically active provirus with replicative capacities has not yet been demonstrated. To confirm the assumption that HERV-K codes for the previously observed retrovirus-like particles (human teratocarcinoma-derived virus, HTDV) in human teratocarcinoma cells, we have constructed recombinant full-length HERV-K cDNA-based baculoviruses withgag, pro, pol,andenvORFs. Two viral constructs were used for infections of insect cells, one bearing 67 bp of the 5′ untranslated region upstream of the 5′ splice donor (SD) site and of the retroviral genes, the second omitting the SD sequence. For both recombinant viruses, indirect immunofluorescence and laser scan analyses revealed expression of HERV-K Gag protein. Electron microscopy studies demonstrated efficient production of virus-like particles (VLPs) at the cytoplasmic cell membranes. These VLPs are morphologically identical with the HTDV phenotype. In immunoelectron microscopy of ultrathin frozen sections, anti-HERV-K Gag antibodies specifically reacted with HERV-K VLPs. In Western blots, in addition to the 76-kDa precursor protein, the putative major core protein with an apparent molecular mass of 32 kDa exhibited predominant immunoreactivity with anti-Gag antiserum. In contrast, neither HERV-K Env nor cORF proteins could be detected due to inefficient mRNA splicing. Purified particles from insect cell culture supernatants tested in an ultrasensitive reverse transcriptase assay revealed weak polymerase activity. The data demonstrate that HERV-K codes for retroviral particles of the HTDV phenotype.