Porcine Endogenous Retrovirus RNA Research Articles

Evidence for Microchimerism in Baboon Recipients of Pig Hearts.

Xenotransplantation, like allotransplantation, is usually associated with microchimerism, i.e., the presence of cells from the donor in the recipient. Microchimerism was reported in first xenotransplantation trials in humans, as well as in most preclinical trials in nonhuman primates (for review, see Denner, Viruses 2023, 15, 190). When using pigs as xenotransplantation donors, their cells contain porcine endogenous retroviruses (PERVs) in their genome. This makes it difficult to discriminate between microchimerism and PERV infection of the recipient. Here, we demonstrate the appropriate virological methods to be used for the identification of microchimerism, first by screening for porcine cellular genes, and then how to detect infection of the host. Using porcine short interspersed nuclear sequences (SINEs), which have hundreds of thousands of copies in the pig genome, significantly increased the sensitivity of the screening for pig cells. Second, absence of PERV RNA demonstrated an absence of viral genomic RNA or expression as mRNA. Lastly, absence of antibodies against PERV proteins conclusively demonstrated an absence of a PERV infection. When applying these methods for analyzing baboons after pig heart transplantation, microchimerism could be demonstrated and infection excluded in all animals. These methods can be used in future clinical trials.

Assessment of porcine endogenous retrovirus transmission across an alginate barrier used for the encapsulation of porcine islets

Subcutaneous implantation of a macroencapsulated patch containing human allogenic islets has been successfully used to alleviate type 1 diabetes mellitus (T1DM) in a human recipient without the need for immunosuppression. The use of encapsulated porcine islets to treat T1DM has also been reported. Although no evidence of pathogen transfer using this technology has been reported to date, we deemed it appropriate to determine if the encapsulation technology would prevent the release of virus, in particular, the porcine endogenous retrovirus (PERV). HEK293 (human epithelial kidney) and swine testis (ST) cells were co-cultured with macroencapsulated pig islets embedded in an alginate patch, macroencapsulated PK15 (swine kidney epithelial) cells embedded in an alginate patch and free PK15 cells. Cells and supernatant were harvested at weekly time points from the cultures for up to 60days and screened for evidence of PERV release using qRT-PCR to detect PERV RNA and SG-PERT to detect reverse transcriptase (RT). No PERV virus, or evidence of PERV replication, was detected in the culture medium of HEK293 or pig cells cultured with encapsulated porcine islets. Increased PERV activity relative to the background was not detected in ST cells cultured with encapsulated PK15 cells. However, PERV was detected in 1 of the 3 experimental replicates of HEK293 cells cultured with encapsulated PK15 cells. Both HEK293 and ST cells cultured with free PK15 cells showed an increase in RT detection. With the exception of 1 replicate, there does not appear to be evidence of transmission of replication competent PERV from the encapsulated islet cells or the positive control PK15 cells across the alginate barrier. The detection of PERV would suggest the alginate barrier of this replicate may have become compromised, emphasizing the importance of quality control when producing encapsulated islet patches.

Corneal Xenotransplantation From Pig to Rhesus Monkey: No Signs of Transmission of Endogenous Porcine Retroviruses

BackgroundXenotransplantations of pig corneas have become an attractive alternative to human corneas. Such xenotransplantations carry the danger, however, of transmission of porcine endogenous retroviruses (PERVs). Here, we investigated whether porcine corneas harbor viral DNA and RNA and whether transplantation to a nonhuman primate would lead to host PERV infection. MethodsMonkey vein endothelial cells (MVECs) were inoculated with porcine aortic endothelial cell (PAEC) supernatants, and DNA and total RNA of MVECs were tested for PERV by polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) assays. Corneas were harvested from Wuzhishan miniature pigs, and the presence of PERV proviral DNA and RNA was analyzed by PCR and RT-PCR, respectively. Fresh or dehydrated corneas were then transplanted to rhesus monkeys, and PERV proviral DNA and RNA were analyzed in host peripheral blood lymphocytes at 6 and 24 months. Furthermore, the presence of PERV sequences was analyzed in the transplant at 24 months. ResultsPCR analysis showed PERV transfection from PAECs to MVECs in vitro. PCR and RT-PCR gave positive signals for PERV subtypes A and B, but not PERV-C, regardless of how the corneas were prepared. No evidence was found for PERV transmission to the host, and the transplant had lost its viral signal at the end of the 24-month period. ConclusionsRegardless of cornea preparation and storage, PERV transmission from pig to host could not be detected, despite that the transplant was initially PERV-positive. The use of the Wuzhishan miniature pig as the donor may be advantageous because it lacks PERV-C and hence potentially infectious A/C recombinants.

Long-term safety from transmission of porcine endogenous retrovirus after pig-to-non-human primate corneal transplantation.

The risk of xenozoonosis mainly by porcine endogenous retrovirus (PERV) has been considered as one of the main hurdles in xenotransplantation and therefore should be elucidated prior to the clinical use of porcine corneal grafts. Accordingly, an investigation was performed to analyze the infectivity of PERVs from porcine keratocytes to human cells, and the long-term risk of transmission of PERVs was determined using pig-to-non-human primate (NHP) corneal transplantation models. The infectivity of PERVs from the SNU miniature pig keratocytes was investigated by coculture with a human embryonic kidney cell line. Twenty-two rhesus macaques underwent xenocorneal transplantation as follows: (i) group 1 (n=4): anterior lamellar keratoplasty (LKP) with freshly preserved porcine corneas, (ii) group 2 (n=5): anterior LKP with decellularized porcine corneas followed by penetrating keratoplasty (PKP) with allografts, (iii) group 3 (n=3): PKP under steroid-based immunosuppression, (iv) group 4 (n=4): PKP under anti-CD154 antibody-based immunosuppression, (v) group 5 (n=4): deep anterior LKP with freshly preserved porcine corneas under anti-CD40 antibody-based immunosuppression, and (vi) group 6 (n=2): PKP under anti-CD40 antibody-based immunosuppression. Postoperative blood samples were serially collected, and tissue samples were obtained from thirteen different organs at the end of each experiment. The existence of PERV DNA and RNA was investigated using PCR and RT-PCR. Using two independent in vitro infectivity tests, neither PERV pol nor pig mitochondrial cytochrome oxidase II was detected after 41 and 92days of coculture, respectively. After xenocorneal transplantation, a total of 257 serial peripheral blood mononuclear cell samples, 34 serial plasma samples, and 282 tissue samples were obtained from the NHP recipients up to 1176days post-transplantation. No PERV transmission was evident in any samples. Within the limits of this study, there is no evidence to support any risk of PERV transmission from porcine corneal tissues to NHP recipients, despite the existence of PERV-expressing cells in porcine corneas.

No PERV transmission during a clinical trial of pig islet cell transplantation

Xenotransplantation of pig islet cells is a promising alternative for the treatment of diabetes with insulin and may help to prevent numerous late complications such as blindness and amputation. First encouraging results using porcine islets have been reported in preclinical animal models as well in the first clinical trial in New Zealand. The goal of this manuscript is to examine the biological safety of a second trial performed in Argentina, specifically in regards to the transmission of porcine endogenous retroviruses (PERVs) using improved detection methods As in the first trial encapsulated islet cells from the well-characterised Auckland Island pigs were used. The animals were not genetically modified. The islet cells were transplanted in eight human recipients using a modified clinical protocol. Sera taken at different time points after transplantation (up to 55 weeks) were screened for the presence of antibodies against PERV proteins by Western blot analysis using viral antigens from highly purified virus particles. Positive sera obtained by immunization with recombinant PERV proteins were used as control sera. In none of the patients antibodies against PERV were detected, indicating the absence of infection. In parallel at different time points (up to 113 weeks) white blood cells (WBC) have been tested for PERV DNA, and WBC and plasma for PERV RNA by real-time RT-PCR. All tests were negative. In addition, using primers detecting pig mitochondrial cytochrome oxidase (COX) gene, patients were screened for microchimerism. In summary, the data are further evidence for the safety of pig islet cell transplantation.

No transmission of porcine endogenous retrovirus in an acute liver failure model treated by a novel hybrid bioartificial liver containing porcine hepatocytes

A novel hybrid bioartificial liver (HBAL) was constructed using an anionic resin adsorption column and a multi-layer flat-plate bioreactor containing porcine hepatocytes co-cultured with bone marrow mesenchymal stem cells (MSCs). This study aimed to evaluate the microbiological safety of the HBAL by detecting the transmission of porcine endogenous retroviruses (PERVs) into canines with acute liver failure (ALF) undergoing HBAL. Eight dogs with ALF received a 6-hour HBAL treatment on the first day after the modeling by D-galactosamine administration. The plasma in the HBAL and the whole blood in the dogs were collected for PERV detection at regular intervals until one year later when the dogs were sacrificed to retrieve the tissues of several organs for immunohistochemistry and Western blotting for the investigation of PERV capsid protein gag p30 in the tissue. Furthermore, HEK293 cells were incubated to determine the in vitro infectivity. PERV RNA and reverse transcriptase activity were observed in the plasma of circuit 3, suggesting that PERV particles released in circuit 3. No positive PERV RNA and reverse transcriptase activity were detected in other plasma. No HEK293 cells were infected by the plasma in vitro. In addition, all PERV-related analyses in peripheral blood mononuclear cells and tissues were negative. No transmission of PERVs into ALF canines suggested a reliable microbiological safety of HBAL based on porcine hepatocytes.

Screening pigs for xenotransplantation: expression of porcine endogenous retroviruses in transgenic pig skin.

Pigs seem to be the answer to worldwide organ donor shortage. Porcine skin may also be applied as a dressing for severe burns. Genetic modifications of donor animals enable reduction of immune response, which prolongs xenograft survival as temporary biological dressing and allows achieving resistance against xenograft rejection. The risk posed by porcine endogenous retroviruses (PERVs) cannot be eliminated by breeding animals under specific-pathogen-free conditions and so all recipients of porcine graft will be exposed to PERVs. Therefore our study has been focused on the assessment of PERV DNA and mRNA level in skin samples of transgenic pigs generated for xenotransplantation. Porcine skin fragments were obtained from 3- to 6-month-old non-transgenic and transgenic Polish Landrace pigs. Transgenic pigs were produced by pronuclear DNA microinjection and were developed to express the human α-galactosidase and the human α-1,2-fucosyltransferase gene. The copy numbers of PERV DNA and RNA were evaluated using real-time Q-PCR and QRT-PCR. Comparative analysis of all PERV subtypes revealed that PERV-A is the main subtype of PERVs in analyzed skin samples. There was no significantly different copy number of PERV-A, PERV-B and PERV-C between non-transgenic pigs, pigs with the human α-galactosidase and pigs expressing the human α-1,2-fucosyltransferase gene, except of PERV-C DNA. It brings the conclusion, that transgenesis process exerts no influence on PERVs transinfection. That is another step forward in the development of pig skin xenografts as burn wounds dressing.

Quantitative Analysis of Porcine Endogenous Retroviruses in Different Organs of Transgenic Pigs Generated for Xenotransplantation

The pig appears to be the most promising animal donor of organs for use in human recipients. Among several types of pathogens found in pigs, one of the greatest problems is presented by porcine endogenous retroviruses (PERVs). Screening of the source pig herd for PERVs should include analysis of both PERV DNA and RNA. Therefore, the present study focuses on quantitative analysis of PERVs in different organs such as the skin, heart, muscle, and liver and blood of transgenic pigs generated for xenotransplantation. Transgenic pigs were developed to express the human α-galactosidase, the human α-1,2-fucosyltransferase gene, or both genetic modifications of the genome (Lipinski et al., Medycyna Wet 66:316-322, 2010; Lipinski et al., Ann Anim Sci 12:349-356, 2012; Wieczorek et al., Medycyna Wet 67:462-466, 2011). The copy numbers of PERV DNA and RNA were evaluated using real-time Q-PCR and QRT-PCR, respectively. Comparative analysis of all PERV subtypes revealed the following relationships: PERV A>PERV B>PERV C. PERV A and B were found in all samples, whereas PERV C was detected in 47% of the tested animals. The lowest level of PERV DNA was shown in the muscles for PERV A and B and in blood samples for PERV C. The lowest level of PERV A RNA was found in the skin, whereas those of PERV B and C RNA were found in liver specimens. Quantitative analysis revealed differences in the copy number of PERV subtypes between various organs of transgenic pigs generated for xenotransplantation. Our data support the idea that careful pig selection for organ donation with low PERV copy number may limit the risk of retrovirus transmission to the human recipients.

Microbiological safety of a novel bio-artificial liver support system based on porcine hepatocytes: a experimental study

BackgroundOur institute has developed a novel bio-artificial liver (BAL) support system, based on a multi-layer radial-flow bioreactor carrying porcine hepatocytes and mesenchymal stem cells. It has been shown that porcine hepatocytes are capable of carrying infectious porcine endogenous retroviruses (PERVs) into human cells, thus the microbiological safety of any such system must be confirmed before clinical trials can be performed. In this study, we focused on assessing the status of PERV infection in beagles treated with the novel BAL.MethodsFive normal beagles were treated with the novel BAL for 6 hours. The study was conducted for 6 months, during which plasma was collected from the BAL and whole blood from the beagles at regular intervals. DNA and RNA in both the collected peripheral blood mononuclear cells (PBMCs) and plasma samples were extracted for conventional PCR and reverse transcriptase (RT)-PCR with PERV-specific primers and the porcine-specific primer Sus scrofa cytochrome B. Meanwhile, the RT activity and the in vitro infectivity of the plasma were measured.ResultsPositive PERV RNA and RT activity were detected only in the plasma samples taken from the third circuit of the BAL system. All other samples including PBMCs and other plasma samples were negative for PERV RNA, PERV DNA, and RT activity. In the in vitro infection experiment, no infection was found in HEK293 cells treated with plasma.ConclusionsNo infective PERV was detected in the experimental animals, thus the novel BAL had a reliable microbiological safety profile.

Factors Influencing the Transfer of Porcine Endogenous Retroviruses across the Membrane in Bioartificial Livers

to investigate the factors influencing the transfer of porcine endogenous retroviruses (PERVs) across the membrane in a new bioartificial liver (BAL). A new BAL containing 2 circuits was constructed using plasma component separators with membrane pore sizes of 10 nm, 20 nm, 30 nm, and 35 nm, or a plasma filter with a membrane pore size of 500 nm. Cocultured cells of porcine hepatocytes and mesenchymal stem cells or single porcine hepatocytes were incubated in the bioreactors, and the BAL worked for 72 hours, with supernatant samples in internal and external circuits collected every 12 hours. PERV RNA, reverse transcriptase (RT) activity, and in vitro infectivity of the supernatant were detected. With the plasma filters, the results of PERV detection were the same in both circuits. With plasma component separators, PERV RNA was found in the external circuits, but no positive RT activity or HEK293 cell infection was found. The time at which the PERV RNA was first detected varied with the pore size of membrane; the larger the membrane pore size was, the earlier the RNA was detected. The PERV RNA level in the external circuits was reduced significantly compared with that in the internal circuits at any detecting time. The plasma component separators with membrane pore size =35 nm could significantly reduce the passage of infectious PERVs. And the membrane pore size, the treatment duration, and the viral level in the internal circuit were potential factors influencing the transfer of PERVs across the membrane in a BAL. In addition, a low risk of PERV transmission from porcine hepatocytes to human cells was found.

Evaluation of a novel hybrid bioartificial liver based on a multi-layer flat-plate bioreactor

To evaluate the efficacy and safety of a hybrid bioartificial liver (HBAL) system in the treatment of acute liver failure. Canine models with acute liver failure were introduced with intravenous administration of D-galactosamine. The animals were divided into: the HBAL treatment group (n = 8), in which the canines received a 3-h treatment of HBAL; the bioartificial liver (BAL) treatment group (n = 8), in which the canines received a 3-h treatment of BAL; the non-bioartificial liver (NBAL) treatment group (n = 8), in which the canines received a 3-h treatment of NBAL; the control group (n = 8), in which the canines received no additional treatment. Biochemical parameters and survival time were determined. Levels of xenoantibodies, RNA of porcine endogenous retrovirus (PERV) and reverse transcriptase (RT) activity in the plasma were detected. Biochemical parameters were significantly decreased in all treatment groups. The TBIL level in the HBAL group was lower than that in other groups (2.19 ± 0.55 μmol/L vs 24.2 ± 6.45 μmol/L, 12.47 ± 3.62 μmol/L, 3.77 ± 1.83 μmol/L, P < 0.05). The prothrombin time (PT) in the BAL and HBAL groups was significantly shorter than the NBAL and control groups (18.47 ± 4.41 s, 15.5 ± 1.56 s vs 28.67 ± 5.71 s, 21.71 ± 3.4 s, P < 0.05), and the PT in the HBAL group was shortest of all the groups. The albumin in the BAL and HBAL groups significantly increased and a significantly higher level was observed in the HBAL group compared with the BAL group (27.7 ± 1.7 g/L vs 25.24 ± 1.93 g/L). In the HBAL group, the ammonia levels significantly decreased from 54.37 ± 6.86 to 37.75 ± 6.09 after treatment (P < 0.05); there were significant difference in ammonia levels between other the groups (P < 0.05). The levels of antibodies were similar before and after treatment. The PERV RNA and the RT activity in the canine plasma were all negative. The HBAL showed great efficiency and safety in the treatment of acute liver failure.

Transmission of zoonoses in xenotransplantation: Porcine endogenous retroviruses from an immunological and molecular point of view

Pigs offer an unlimited source of xenografts for humans. The use of transplants from animal origin offers a potential solution to the limited supply of human organs and tissues. However, like many other mammalian species, pigs harbor porcine endogenous retrovirus (PERV), which are encoded in their genomic DNA and are assumed to have been integrated into the porcine germ line more than 7.6 × 106 years ago and showing that the age correlates with the time of separation between pigs and peccaries 7.4 × 106 years ago. The ability of the PERV to infect human cells in vitro has heightened safety concerns regarding the transmission of PERV to pig xenograft recipients. In this study, we detected PERV-AC recombinant virus in porcine genomic DNA that may have resulted from exogenous viral recombination. Infectious risk in xenotransplantation will be defined by the activity of PERV loci in vivo. We identified unique Haemophilus aegyptius III HaeIII gag restriction fragment length polymorphism (RFLP) profiles resulting from single nucleotide polymorphisms; these were found only in animals that produced human tropic PERV. Porcine tissues were analyzed using validated assays specific for PERV: polymerase chain reaction (PCR) (for PERV DNA), RT-PCR (for PERV RNA), cell culture, RFLP analysis, and sequence analysis. These findings have demonstrated that the presence of both DNA and RNA forms of porcine endogenous retrovirus in porcine tissues needs to be carefully considered when the infectious disease potential of xenotransplantation is being assessed.

New evidence of porcine endogenous retrovirus transmission with new bio-artificial liver system: a experimental study

To investigate the potential transmissibility of porcine endogenous retrovirus (PERV) from a newly-developed porcine hepatocyte bioartificial liver (BAL) system prior to human clinical trial by using a live canine model. Five normal beagles were treated with the new BAL support system for six hours. Samples of plasma from the BAL system and whole blood from the beagles were collected at regular intervals over the six month study period. DNA and RNA were isolated from both the peripheral blood mononuclear cells (PBMCs) and plasma for evaluation by polymerase chain reaction (PCR) and reverse transcription (RT)-PCR, respectively, to detect PERV and the Sus scrofa cytochrome B normalization standard. In addition, RT activity and the in vitro infectivity of the plasma were detected in HEK293 cells. All five beagles remained in stable physical health throughout the treatment and survived until the end of the study. PERV RNA-positivity and RT activity were only detected in the plasma samples from the 3rd BAL treatment cycle. All other samples, including PBMCs and plasma, were negative for PERV RNA, PERV DNA, and RT activity. In addition, none of the sera samples showed in vitro infectivity. Application of our BAL system does not lead to PERV transmission.

Influence of chitosan nanofiber scaffold on porcine endogenous retroviral expression and infectivity in pig hepatocytes

To investigate the influence of chitosan nanofiber scaffold on the production and infectivity of porcine endogenous retrovirus (PERV) expressed by porcine hepatocytes. Freshly isolated porcine hepatocytes were cultured with or without chitosan nanofiber scaffold (defined as Nano group and Hep group) for 7 d. The daily collection of culture medium was used to detect reverse transcriptase (RT) activity with RT activity assay kits and PERV RNA by reverse transcription-polymerase chain reaction (PCR) and real time PCR with the PERV specific primers. And Western blotting was performed with the lysates of daily retrieved cells to determine the PERV protein gag p30. Besides, the in-vitro infectivity of the supernatant was tested by incubating the human embryo kidney 293 (HEK293) cells. The similar changing trends between two groups were observed in real time PCR, RT activity assay and Western blotting. Two peaks of PERV expression at 10H and Day 2 were found and followed by a regular decline. No significant difference was found between two groups except the significantly high level of PERV RNA at Day 6 and PERV protein at Day 5 in Nano group than that in Hep group. And in the in-vitro infection experiment, no HEK293 cell was infected by the supernatant. Chitosan nanofiber scaffold might prolong the PERV secreting time in pig hepatocytes but would not obviously influence its productive amount and infectivity, so it could be applied in the bioartificial liver without the increased risk of the virus transmission.

Long-term absence of porcine endogenous retrovirus infection in chronically immunosuppressed patients after treatment with the porcine cell-based Academic Medical Center bioartificial liver

Clinical use of porcine cell-based bioartificial liver (BAL) support in acute liver failure as bridging therapy for liver transplantation exposes the patient to the risk of transmission of porcine endogenous retroviruses (PERVs) to human. This risk may be enhanced when patients receive liver transplant and are subsequently immunosuppressed. As further follow-up of previously reported patients (Di Nicuolo et al. 2005), an assessment of PERV infection was made in the same patient population pharmacologically immunosuppressed for several years after BAL treatment and in healthcare workers (HCWs) involved in the clinical trial at that time. Plasma and peripheral blood mononuclear cells (PBMCs) from eight patients treated with the Academic Medical Center-BAL (AMC-BAL), who survived to transplant, and 13 HCWs, who were involved in the trial, were assessed to detect PERV infection. A novel quantitative real-time polymerase chain reaction assay has been used. Eight patients who received a liver transplant after AMC-BAL treatment are still alive under long-term pharmacological immunosuppression. The current clinical follow-up ranges from 5.6 to 8.7 yr after BAL treatment. A new q-real-time PCR assay has been developed and validated to detect PERV infection. The limit of quantification of PERV DNA was ≥ 5 copies per 1 × 10(5) PBMCs. The linear dynamic range was from 5 × 10(0) to 5 × 10(6) copies. In both patients and HCWs, neither PERV DNA in PBMCs nor PERV RNA in plasma and PBMC samples have been found. Up to 8.7 yr after exposure to treatment with porcine liver cell-based BAL, no PERV infection has been found in long-term immunosuppressed patients and in HCWs by a new highly sensitive and specific q-real-time PCR assay.

Polymerase chain reaction in detection of porcine endogenous retrovirus (PERV) from porcine tissues

Pigs offer an unlimited source of xenografts for humans. The use of transplants from animal origin offers a potential solution to the limited supply of human organs and tissues. However, like many other mammalian species, pigs harbor porcine endogenous retrovirus (PERV), which are encoded in their genomic DNA and are assumed to have been integrated into the porcine germline. The ability of PERV to infect human cells in vitro has heightened safety concerns regarding the transmission of PERV to pig xenograft recipients. Porcine tissues were analyzed using validated assays specifi c for PERV: polymerase chain reaction (PCR) (for PERV DNA) and reverse transcriptase (RT)-PCR (for PERV RNA). PERV-specifi c gag sequences were found in the porcine heart tissue samples using DNA-PCR and RT-PCR. PCR is a rapid and specifi c test for the detection of PERV from xenografts. These fi ndings have demonstrated that the presence of both DNA and RNA forms of PERV in porcine tissues needs to be carefully considered when the infectious disease potential of xenotransplantation is being assessed.

Porcine Endogenous Retrovirus (PERV) and its Transmission Characteristics: A Study of the New Zealand Designated Pathogen-Free Herd

Previously a strategy for monitoring of pigs intended for cell transplantation was developed and successfully applied to several representative herds in New Zealand. A designated pathogen-free (DPF) herd has been chosen as a good candidate for xenotransplantation. This herd has previously tested free of infectious agents relevant to xenotransplantation and we present here an in depth study of porcine endogenous retrovirus (PERV) transmission. A panel of assays that describes the constraints for the transmission of PERV has been suggested. It includes a) infectivity test in coculture of DPF pig primary cells with both human and pig target cell lines; b) RT activity in supernatant of stimulated primary cells from DPF pigs; c) viral load in donor's blood plasma; d) PERV proviral copy number in DPF pig genome; e) PERV class C prevalence in the herd and its recombination potential. There was no evidence of PERV transmission from DPF pig tissue to either pig or human cells. Additionally, there was no evidence of PERV RNA present in pig blood plasma. PERV copy number differs in individual pigs from as low as 3 copies to 30 copies and the presence of PERV-C varied between animals and breeds. In all DPF pigs tested, a specific locus for PERV-C potentially associated with the recombination of PERV in miniature swine was absent. Presented data on the PERV transmission allows us to classify the DPF potential donors as "null" or noninfectious pigs.

Lack of evidence for PERV expression after apoptosis‐mediated horizontal gene transfer between porcine and human cells

Evidence for porcine endogenous retrovirus (PERV) infection of human cells has provoked a public health debate over the proposed use of porcine xenografts to alleviate the worldwide shortage of human allografts. Nevertheless, the potential relevance of PERV transmission by apoptosis-mediated horizontal DNA transfer, a documented means of infection-independent retrovirus delivery, appears to have been overlooked in this discussion. To examine the hypothesis that apoptotic cell death during porcine xenograft rejection is capable of fostering horizontal DNA transfer, we have now assessed in vitro cocultures, consisting of phagocytic human fibroblasts and apoptotic or necrotic porcine B-lymphoblastoid cells, for evidence of cross-species PERV exchange and eventual replication. Using real-time polymerase chain reaction (PCR) assays, designed to differentiate nuclear and cytoplasmic DNA derived from either porcine or human cells, we now report evidence for the presence of porcine DNA, including PERV, in the nucleus of human fibroblasts exposed to apoptotic porcine cells. This novel demonstration of apoptosis-mediated horizontal PERV transfer is characterized by a low efficiency of transfer and a transient nature, being present in only 0.22% of the cocultured human cells and disappearing to undetectable levels within 4 weeks of exposure to apoptotic porcine cells. In contrast, using PERV-specific real-time reverse-transcriptase PCR (RT-PCR) and ultra-sensitive product-enhanced reverse transcriptase (PERT) assays, we find no evidence for human fibroblast-derived cellular PERV RNA or coculture supernatant-based RT-activity, indicating a lack of subsequent PERV replication. Together, these results suggest that apoptosis-mediated horizontal PERV transfer does not present an overt hazard within the framework of porcine xenotransplantation. However, we also present arguments against extrapolation of these in vitro observations directly to clinical circumstances.

Retroviremia in Commercial Pigs and Its Preliminary Association with Poor Health

Retroviremia in Commercial Pigs and Its Preliminary Association with Poor Health

Porcine Endogenous Retrovirus Encodes Xenoantigens Involved in Porcine Cellular Xenograft Rejection by Mice

Identification of the antigens that stimulate transplant rejection can help develop graft-specific antirejection strategies. The xenoantigens recognized during rejection of porcine cellular xenografts have not been clearly defined, but it has been assumed that major histocompatibility complex (MHC) xenoantigens are involved. The role of porcine endogenous retrovirus (PERV) as a source of xenoantigens was examined. The authors used morphometry to compare the kinetics of swine leukocyte antigen (SLA) pig thyroid xenograft rejection in control mice and mice immunized with PERV PK15 cells (porcine kidney epithelial cells), PERV SLA pig peripheral blood lymphocytes (PBL), PERV virions purified from PK15 cells, and PERV or PERV A pseudotypes produced from infected human 293 cells. The tempo of rejection for cellular xenografts of PERV A pseudotype-producing human 293 cells, uninfected human 293 cells, and PK15 cells in PERV-preimmunized and control mice was also compared. Mice immunized with PK15 cells rejected pig thyroid xenografts significantly faster at day 5 than control mice and mice immunized with pig PBL. This correlated with the amount of PERV RNA and virions produced, but not with the amount of SLA class I MHC expressed by PK15 cells. Immunization of mice with PERV virions purified from porcine PK15 cells and with PERV virions or PERV A pseudotypes produced by human 293 cells also induced accelerated xenograft rejection by 5 days. Accelerated rejection induced by virus pretreatment was CD4 T-cell dependent and restricted to PERV-expressing cellular xenografts of porcine or nonporcine origin. PERV acts as a significant source of xenoantigens that target porcine cellular xenografts for rejection.