Porcine Endogenous Retrovirus Infection Research Articles

Microarray Analysis of Retroviral Restriction Factor Gene Expression in Response to Porcine Endogenous Retrovirus Infection

Microarray analysis has been used for screening genes involved in specific biological processes. Many studies have shown that restriction factors may play an important role in xenotransplantation safety, but it is still unclear whether porcine endogenous retroviruses (PERVs) may be inhibited by these factors. Therefore, the present study focused on the microarray analysis retroviral restriction factors gene expression in normal human dermal fibroblasts (NHDFs) in response to PERVs. PERV infectivity was analyzed using a co-culture system of NHDFs and porcine kidney epithelial cells (PK15 cell line). Detection of the copy number of PERV A, PERV B DNA and PERV A, PERV B RNA was performed using real-time Q-PCR and QRT-PCR. The expression of retroviral restriction factor genes was compared between PERV-infected and uninfected NHDF cells using oligonucleotide microarray. The up-regulated transcripts were recorded for two differentially expressed genes (TRIM1, TRIM16) with the use of GeneSpring platform and Significance Analysis of Microarrays. In conclusion, our results suggest that the TRIM family may play an important role in innate immunity to PERV infection. These results can allow a better understanding of restriction mechanism of PERV infection and probably design molecularly targeted therapies in the future. Moreover, knowledge of retroviral restriction factor gene expression in human cells may help to uncover strategies for determining their exact function. Microarray analyses seem to be promising in biological and biomedical studies, however, these results should be further confirmed by research conducted at the protein level.

Differential Expression of Tripartite Motif-Containing Family in Normal Human Dermal Fibroblasts in Response to Porcine Endogenous Retrovirus Infection

Antiretroviral restriction factors may play an essential role in the safety of xenotransplantation. Therefore, the present study focused on investigation of the changes in the tripartite motif-containing family (TRIM) gene expression in normal human dermal fibroblasts with and without lipopolysaccharide stimulation in response to porcine endogenous retrovirus infection. Analysis of the expression profile of TRIMs was performed using oligonucleotide microarrays and QRT-PCR. Nine (TRIM1, TRIM2, TRIM5, TRIM14, TRIM16, TRIM18, TRIM22, TRIM27 and TRIM31) statistically significantly differentially expressed genes were found (P < 0.05, one-way ANOVA). In conclusion, comprehensive analysis of retroviral restriction factor gene expression in human dermal fibroblasts before and after porcine endogenous retrovirus infection with and without LPS stimulation may suggest association of the selected TRIMs with antiretroviral activity.

Susceptibility of Human Liver Cells to Porcine Endogenous Retrovirus

The risk of porcine endogenous retrovirus infection is a major barrier for pig-to-human xenotransplant. Porcine endogenous retrovirus, present in porcine cells, can infect many human and nonhuman primate cells in vitro, but there is no evidence available about in vitro infection of human liver cells. We investigated the susceptibility of different human liver cells to porcine endogenous retrovirus. The supernatant from a porcine kidney cell line was added to human liver cells, including a normal hepatocyte cell line (HL-7702 cells), primary hepatocytes (Phh cells), and a liver stellate cell line (Lx-2 cells), and to human embryonic kidney cells as a reference control. Expression of the porcine endogenous retrovirus antigen p15E in the human cells was evaluated with polymerase chain reaction, reverse transcription-polymerase chain reaction, and Western blot. The porcine endogenous retrovirus antigen p15E was not expressed in any human liver cells (HL-7702, Phh, or Lx-2 cells) that had been exposed to supernatants from porcine kidney cell lines. Porcine endogenous retrovirus-specific fragments were amplified in human kidney cells. Human liver cells tested were not susceptible to infection by porcine endogenous retrovirus. Therefore, not all human cells are susceptible to porcine endogenous retrovirus.

Knockdown of Porcine Endogenous Retroviruses by RNA Interference in Chinese Experimental Miniature Pig Fibroblasts

BackgroundThe clinical application of porcine-derived xenotransplants is limited by the potential risk of infection due to the presence of porcine endogenous retrovirus (PERV) in tissues, organs, and cells. The establishment of pig fibroblasts with low PERV expression and without PERV-C can provide a nuclear donor to generate a safer transgenic pig. MethodsIn this study, we obtained Chinese Experimental Miniature Pig fibroblasts (CEMPF) with low expression of PERV and none of PERV-C. We designed small interfering RNA (siRNA) expressed as short hairpin RNAs (shRNA) based on the highly conserved gag and pol regions of PERV and screened for the most effective siRNA to inhibit PERV expression. The selected shRNA-pol3 fragment was introduced into the CEMPF to obtain an engineered CEMPF stably expressing shRNA-pol3. ResultsThe PERV mRNA expression level in the engineered CEMPF was only 7.9% of that observed in fibroblasts from wild-type CEMPF, PERV P15E protein expression was significantly reduced. HEK293 cells cocultured with the supernate of the engineered CEMPF showed no PERV infection. ConclusionsEngineered CEMPF, which possess no risk of PERV-A/C infection, can serve as a nuclear donor to generate xenograft donor pigs.

Porcine endogenous retrovirus infection changes the expression of inflammation-related genes in lipopolysaccharide-stimulated human dermal fibroblasts

The present study focuses on explaining the interaction between porcine endogenous retroviruses (PERVs) and human cells in inflammatory conditions. The differences in expression of selected inflammation-related genes in human dermal fibroblasts (NHDF) infected with PERVs with and without lipopolysaccharide stimulation were identified. The PERV infectivity was analyzed using a co-culture of NHDF and PK15 cells. Quantification of PERV A, B DNA and PERV A, B RNA was performed by real-time QPCR and QRT-PCR. The analysis of the expression profile was performed using HG-U133A 2.0 oligonucleotide microarrays. PERV infection of NHDF cells with LPS stimulation resulted in a statistically significant decrease in the copy number of PERV A DNA, and an increase in the copy number of PERV A RNA compared to fibroblasts without stimulation. There was no statistically significant difference between the copy number of PERV B RNA of LPStreated and untreated NHDF cells. Typing of differentiation genes was performed in a panel of 571 selected transcripts of inflammation-related genes. Among all studied genes, 23 were differentially regulated with a change greater that 1.1-fold and p<0.05 in all studied groups. Of these 23 genes, 3 were found to be regulated by more than 2.0-fold at least in 2 studied groups (IL6, IL8, and IL33). The interaction between porcine endogenous retroviruses and human cells changes in inflammatory conditions. PERV infection of NHDF cells may alter the expression of inflammation-related genes. Further investigations concerning PERV infection of human cells in different conditions seem to be necessary.

Microbiological safety of xenotransplantation compared with allotransplantation

Transmission of viral, bacterial, parasitic, and fungal infections via organ allografts is uncommon but may be associated with life‐threatening disease. Internationally, programs for screening of human organ donors for infectious risk are non‐uniform and vary with national standards and the availability of screening assays. Further, the failure to recognize and/or to report transmission events limits the utility of available data regarding the incidence of allograft‐associated disease transmission. Advances in xenotransplantation biology have allowed some limited clinical trials with the prospect for increased opportunities for clinical xenotransplantation. As with human allotransplantation, the examination of infectious risk has been a central theme in these studies. Significant advances have been made in the breeding and screening of swine for preclinical studies including the identification of novel, potential human pathogens derived from source animals. Thus far, “expected” xenograft‐derived pathogens such as porcine cytomegalovirus (PCMV) have become activated in immunosuppressed primates but have not resulted in systemic infection outside the xenograft. PCMV has been bred out of swine herds by early weaning strategies. Conversely, host pathogens such as primate‐derived cytomegalovirus (CMV) have become activated and have produced serious infectious complications. These infections are preventable using antiviral prophylaxis. Xenogeneic tissues appear to be relatively resistant to infection by common human pathogens such as HIV, HTLV and the hepatitis viruses.Concerns regarding the potential activation of latent porcine retroviruses from xenograft tissues have resulted in the development of novel assays for xenotropic porcine endogenous retrovirus (PERV). PERV transmission to primate xenograft recipients or to human cells in in vivo models has not been detected. Multiple intrinsic cellular mechanisms appear to be active in the prevention of infection of human cells by PERV. Further, PERV appears to be susceptible to available antiretroviral agents. Thus, while the absolute risk for such infections remains unknown in the absence of human studies with prolonged graft survival in immunosuppressed xenograft recipients, the risk of transmission to human recipients appears limited. Some general principles have been developed to guide clinical trials:Outcomes of xenotransplantation trials, including any infectious disease transmissions, should be reported in the scientific literature and to appropriate public health authorities.Surveillance programs should be developed to detect known infectious agents as well as previously unknown or unexpected pathogens in the absence of recognizable clinical syndromes.Standardization of procedures and validation by expert and/or reference laboratories are needed for microbiological assays. Such validation may require international collaboration.Repositories of samples from source animals and from recipients prior to, and following xenograft transplantation are essential to the investigation of possible infectious disease events.Infection is common in allograft recipients. Thus, in advance of clinical trials, policies and procedures should be developed to guide the evaluation of any infectious syndromes that may develop. (e.g. fever of unknown origin [FUO], leukocytosis, leukopenia, graft dysfunction, pneumonia, hepatitis, abscess formation) in xenograft recipients. Based on preclinical experience these procedures will include: (i) Exclusion of infectious syndromes commonly associated with allotransplantation (e.g. CMV, bacterial pneumonia); (ii) Evaluation of PERV infection by serologic and NAT testing; (iii) Assessment of other recipients of xenografts derived from the same herd or source of swine; and (iv) Evaluation of social contacts of the recipient. Consideration of investigation of xenograft recipients for unknown pathogens may require application of advanced research technologies, possibly including use of broad‐range molecular probes, microarrays or high throughput pyrosequencing.

Safety of xenotransplantation: screening the donor pig and the recipient

Introduction: Xenotransplantation using pig cells and tissues may be associated with the transmission of porcine microorganisms including bacteria, parasites, fungi and viruses to the human recipient and may result in zoonones. Porcine endogenous retroviruses (PERVs) represent a special risk since PERV‐A and PERV‐B are present in the genome of all pigs and infect human cells. PERV‐C is not present in all pigs and does not infect human cells. However, recombinants between PERV‐A and PERV‐C have been observed in normal pigs characterised by higher replication rates compared with PERV‐A, and they are also able to infect human cells (1).Methods: In the past years numerous assays based on the PCR technology have been developed to screen for the prevalence and expression of PERV and other porcine microorganisms in the donor pig (2). Whereas most microorganisms may be eliminated by designated pathogen‐free breeding, PERVs cannot be removed this way. In addition, assays have been developed to analyse the recipient for the transmission of PERV and other microorganisms, either using PCR methods or immunological assays to detect an antibody production as a result of infection (3).Results: Using these assays, no transmission of PERV as well as of other porcine microorganisms has been observed in first preclinical and clinical xenotransplantations or animal infection experiments. This was especially true for the first clinical transplantation of pig islet cells approved by the New Zealand government (4). Until now there is no susceptible animal model to study PERV transmission and transplantations of porcine cells or organs to non‐human primates as they are associated with limitations concerning the safety aspect, which do not allow transmitting the negative findings to humans (5). Different experimental approaches are under development to reduce the probability of PERV transmission, e.g. the generation of transgenic pigs expressing PERV‐specific siRNA inhibiting PERV expression by RNA interference (6), genotypic selection of pigs with a low prevalence and expression of PERV and neutralising antibodies against the envelope proteins inhibiting PERV infection (7).Conclusion: Investigations of the last years resulted in highly sensitive and specific methods to study PERV and other microorganisms in donor pigs and human recipients of xenotransplants. These methods showed absence of PERV transmission in all investigated cases, both in more than 200 human xenotransplant recipients, mostly recipients of cellular xenotransplants, as well as in non‐human primates and small animals. New technologies under development may further decrease the probability of transmission.

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.

Porcine factor VIII

Provided are porcine factor VIII nucleotide sequences and hybrid sequences encoding human/porcine chimeric factor VIII-type procoagulant proteins. DNAs encoding such chimeric factor VIII-type procoagulant proteins, pharmaceutical compositions containing such chimeric factor VIII-type procoagulant proteins, and methods of treating hemophilia using such chimeric factor VIII-type procoagulant proteins are also provided.

Infection barriers to successful xenotransplantation focusing on porcine endogenous retroviruses.

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.

Modulation of Porcine Endogenous Retroviruses (PERVs) expression in vitro by shRNA in the presence of cyclosporine A and dexamethasone

Despite the fact that the risk of Porcine Endogenous Retroviruses (PERV) infection and propagation in human recipients is extremely low, such an event cannot be completely ruled out, especially in immunosuppressed patients. Therefore, the aim of this study was to analyze the expression of PERVs in vitro in the presence of immunosuppression agents: cyclosporine A (CsA), and dexamethasone (DEX). We investigated the possible interactions between immunosuppression drugs, CsA and DEX, and the efficiency of anti-PERV RNAi. Plasmid-based vectors expressing shRNAs against all PERV genes were constructed and analyzed. PERVs expression in cultures transfected with anti-PERV RNAi constructions and treated with CsA or DEX was analyzed by Real-Time RT-PCR, Western blot, and by the measurement of RT activity. Both CsA and DEX inhibited PERVs expression in cell cultures in vitro. RNAi constructions efficiently knocked down PERV expression in Circe, and de novo PERV-infected HeLa and HEK-293 cell cultures. Pretreatment of Circe cultures with CsA or DEX increased PERVs knockdown by RNAi, but no specific interaction between the drugs and transfection efficiency was observed. Our results demonstrate that cyclosporine A and dexamethasone decrease expression of PERVs in vitro. We also proved that these drugs did not synergize or antagonize RNAi-mediated knockdown of PERVs. These observations may be beneficial in immunosuppressed xenograft recipients; however, due to the controversial literature data concerning influence of immune suppression on graft recipients, our results should be further analyzed.

Reduction of N-Tropic Mutant Porcine Endogenous Retrovirus Infectivity by Human Tripartite Motif-Containing 5-Isoform Alpha

In cases of retroviral infection, the host cell deploys antiviral proteins as a type of innate immunity. Tripartite motif-containing 5-isoform alpha (TRIM5α) is a potent antiviral protein. TRIM5α has been reported to restrict human immunodeficiency virus (HIV) 1 infection in rhesus monkey cells by targeting the incoming viral capsid at the postentry or preintegration stage of the viral life cycle. As a consequence, virus replication and reverse transcription are interrupted. TRIM5α of human origin has also been shown to inhibit N-tropic murine leukemia virus infection. To investigate the inhibitory effect of TRIM5α on porcine endogenous retrovirus (PERV) infection in humans, we constructed a 293T cell line stably expressing human TRIM5α (293T-huTRIM5α) and tested the infectivity of vesicular stomatitis virus glycoprotein envelope pseudotyped viruses (wild-type PERV [wt-PERV], N-tropic mutant PERV, N-tropic murine leukemia virus, and MoMLV). Infectivity of N-tropic mutant PERV was reduced by 43.3% in 293T-huTRIM5α cells, a decrease in efficiency that was more than 3-fold greater than that of wt-PERV in 293T-huTRIM5α cells. Human TRIM5α exhibited inhibitory activity against N-tropic MLV and N-tropic mutant PERV, but showed no antiviral activity against Moloney murine leukemia virus or wt-PERV.

Multiplex high resolution melting assay for estimation of Porcine Endogenous Retrovirus (PERV) relative gene dosage in pigs and detection of PERV infection in xenograft recipients

Porcine Endogenous Retrovirus (PERV) poses an infectious risk in the field of xenotransplantation. This risk may be mitigated by breeding selectively animals bearing favorable PERV genetic characteristics including pigs with low levels of PERV integrated in the genome. A real-time quantitative polymerase chain reaction (PCR) assay employing the Roche High Resolution Melting (HRM) Master was used to estimate the relative gene dosage of PERV pol integrated within the pig genome. When assessed across 99 pigs of the Auckland Island breed numerous animals bearing low gene dosage were identified. The assay was adapted further to perform multiplex PCR for the detection of PERV infection within xenograft recipients. Besides PERV, amplification targets for the multiplex PCR include a pig cell marker for the determination of microchimerism and an internal amplification control (IAC) to assess the efficiency of nucleic acid isolation and effects of PCR inhibition. When 12 patients who had received porcine islet transplants were tested no evidence of PERV infection was found. The assay was shown to be specific, highly reproducible with superior performance over conventional nested PCR. This assay can be used as both a screening tool for PERV proviral levels within donor pigs and as a diagnostic tool to examine PERV transmission in human patients treated with porcine xenotransplantation material.

Repression of porcine endogenous retrovirus infection by human APOBEC3 proteins

It has been shown that porcine endogenous retrovirus (PERV) can infect human cells, indicating that PERV transmission poses a serious concern in pig-to-human xenotransplantation. A number of recent studies have reported on retrovirus interference by antiviral proteins. The most potent antiviral proteins are members of the APOBEC family of cytidine deaminases, which are involved in defense against retroviral attack. These proteins are present in the cytoplasm of mammalian cells and inhibit retroviral replication. To evaluate the inhibition of PERV transmission by human APOBEC3 proteins, we co-transfected 293T cells with a PERV molecular clone and human APOBEC3F or APOBEC3G expression vectors, and monitored PERV replication competency using a quantitative analysis of PERV pol genes. The replication of PERVs in cells co-expressing human APOBEC3s was reduced by 60–90% compared with PERV-only control. These results suggest that human APOBEC3G and APOBEC3F might serve a potential barrier function against PERV transmission in xenotransplantation.

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.

Long life with or without PERV

Commentary on the original article, Long-term absence of porcine endogenous retrovirus infection…, appearing on pp. 43139

Suboptimal Porcine Endogenous Retrovirus Infection in Non-Human Primate Cells: Implication for Preclinical Xenotransplantation

BackgroundPorcine endogenous retrovirus (PERV) poses a potential risk of zoonotic infection in xenotransplantation. Preclinical transplantation trials using non-human primates (NHP) as recipients of porcine xenografts present the opportunity to assess the zoonosis risk in vivo. However, PERV poorly infects NHP cells for unclear reasons and therefore NHP may represent a suboptimal animal model to assess the risk of PERV zoonoses. We investigated the mechanism responsible for the low efficiency of PERV-A infection in NHP cells.Principal FindingsTwo steps, cell entry and exit, were inefficient for the replication of high-titer, human-tropic A/C recombinant PERV. A restriction factor, tetherin, is likely to be responsible for the block to matured virion release, supported by the correlation between the levels of inhibition and tetherin expression. In rhesus macaque, cynomolgus macaque and baboon the main receptor for PERV entry, PERV-A receptor 1 (PAR-1), was found to be genetically deficient: PAR-1 genes in these species encode serine at amino acid 109 in place of the leucine in human PAR-1. This genetic defect inevitably impacts in vivo sensitivity to PERV infection of these species. In contrast, African green monkey (AGM) PAR-1 is functional, but PERV infection is still poor. Although the mechanism is unclear, tunicamycin treatment, which removes N-glycosylated sugar chains, increases PERV infection, suggesting a possible role for the glycosylation of the receptors.ConclusionsSince cynomolgus macaque and baboon, species often used in pig-to-NHP xenotransplantation experiments, have a defective PAR-1, they hardly represent an ideal animal model to assess the risk of PERV transmission in xenotransplantation. Alternatively, NHP species, like AGM, whose both PARs are functional may represent a better model than baboon and cynomolgus macaque for PERV zoonosis in vivo studies.

No Evidence of the Productive Replication of Porcine Endogenous Retrovirus (PERV) from SNU Miniature Pigs in Human Cell Line

Background: The presence of porcine endogenous retrovirus (PERV) has been considered as one of the main hurdles to transplant pig’s organs or tissues to human beings. There has been no report that PERV infection is associated with human diseases. Because pigs have their own characteristics of PERV according to pig strain, it is necessary to analyze the infectivity of PERV from SNU miniature pig to human cells for future utilization as a transplantation donor. Materials and Methods: Human cell lines were infected with culture supernatant from porcine cell line or immunomodulator-stimulated peripheral blood mononuclear cells (PBMC) of SNU miniature pigs. They were also co-cultured with PBMC or islet cells of SNU miniature pigs. The presence of PERV genes and general pig marker gene in cells was determined by nested PCR with primer set for PERV pol and pig mitochondrial cytochrome oxidase II (COII), respectively. Results: Infection test with the culture supernatant from PBMC of SNU miniature pigs showed that PERV pol but not COII was detected only in a few cases, but there was no uniform infection pattern in scope of stimulators and cell types. PERV pol was not demonstrated in co-cultures of human cell line with PBMC or islet cells from SNU miniature pigs after 80 days of co-cultures. Conclusions: In vitro infectivity test suggests that PERV from SNU miniature pig might not replicate productively in human cell lines although it could infect human cells and integrate into chromosome.

No evidence of porcine endogenous retrovirus in patients with type 1 diabetes after long‐term porcine islet xenotransplantation

Xenotransplantation is a promising alternative for donor shortage to ameliorate physiologic and metabolic disorders. The major concern for xenotransplant is the risk of zoonosis mainly by the porcine endogenous retrovirus (PERV), presentation in the piglet genome. Twenty-three patients with type 1 diabetes were transplanted with porcine islets using collagen-generating devices which were implanted subcutaneously in the anterior wall of the abdomen. Clinical characteristics and metabolic tests were recorded in each visit. They were tested for PERV using PCR and RT-PCR from blood pretransplantation and every 3 months during a 4.6- to 8-year follow-up after their first xenotransplant. Tests by PCR of every DNA sample (780 samples) revealed that there was no PERV infection in the DNA of white cells. No evidence of PERV activation was found in this group of patients with type 1 diabetes during clinical long-term follow-up.