异种移植中猪内源性逆转录病毒的传播

器官移植是治疗各类终末期疾病最有效的手段,为了解决器官移植中供体短缺的问题,人们开始探究异种移植的可能。猪是异种器官移植常见的供体来源之一,猪器官作为连接两个物种间的桥梁,其携带的病毒有可能在物种间传播并存在引起人畜共患病的风险。猪内源性逆转录病毒（PERV）整合在基因组中,是一种跨物种传播的逆转录病毒。本文介绍了PERV传播特性的影响因素、PERV及其重组病毒的传播风险以及异种移植试验中PERV的检测与传播风险评估,以期为缓解器官移植供体严重短缺的现状,推动异种移植技术的发展提供参考。     

Lack of cross-species transmission of porcine endogenous retrovirus (PERV) to transplant recipients and abattoir workers in contact with pigs

This study investigated the potential transmission of porcine endogenous retrovirus (PERV) to solid-organ transplant recipients and abattoir workers in contact with pigs. Blood samples were obtained from volunteer healthy blood donors (Group A; n=33); pig-breeding farmers who had undergone a liver transplant (Group B; n=14); and pig abattoir workers (Group C; n=49). A second blood sample was obtained 1 year after the first sample from 10 of the abattoir workers (Group D). Tests included investigation for PERV-DNA, PERV-RNA, pig-specific mitochondrial DNA, a quantitative detection of PERV nucleic acids, and antibodies to PERV by two different Western Blots. All polymerase chain reaction and Western Blots assays were negative for PERV or antibodies to PERV. Therefore, the risks of cross-species transmission of PERV appear to be negligible for immunocompetent individuals and allotransplant recipients, even if they are in close and repeated contact with live pigs or pig tissues.     

Natural antibodies prevent in vivo transmission of porcine islet-derived endogenous retrovirus to human cells

The discovery of porcine endogenous retroviruses (PERV) has raised concerns regarding the safety of porcine xenotransplantation. However, transmission of PERV had not been observed in humans exposed to porcine tissue. We examined whether PERV derived from porcine pancreatic islet cells could infect human cells in vivo and the role of natural antibodies in inhibiting PERV infection. In vivo infective potential of PERV was studied in SCID mice reconstituted with human peripheral blood leucocytes. Porcine islets were transplanted under the kidney capsule. PERV infection was determined by analyzing PERV gene expression in graft infiltrating lymphocytes (GIL) harvested 21 days posttransplantation. Mice were administered normal human serum prior to and 2 days posttransplantation to study their role in protection of human cells against PERV infection. PERV genes were expressed in all porcine tissues examined, including purified porcine islets. PERV expression was detected in GILs from three of five human-SCID mice. Administration of human serum blocked PERV infection in GILs in five of five human-SCID mice. These results indicate that PERV from porcine islets can infect human cells in vivo. Normal human serum blocks transmission of retrovirus in vivo, suggesting that natural xenoreactive antibodies can prevent PERV infection.     

Analysis of potential porcine endogenous retrovirus (PERV) transmission in a whole-organ xenotransplantation model without interfering microchimerism

The question whether porcine xenografts can lead to porcine endogenous retrovirus (PERV) infection of recipients is critical for the evaluation of the safety of pig-to-man xenotransplantation. Unfortunately, polymerase chain reaction (PCR)-based analysis of potential PERV infections in nonhuman-primate whole-organ xenotransplantation models is hampered by false positive results due to chimeric porcine cells. To avoid the inherent analytical problem of xenomicrochimerism, we developed a non-life-supporting pig-to-primate kidney xenotransplantation model: porcine kidneys were transplanted, whereas the functioning recipient kidneys remained in situ. Subsequent to rejection (after 2 hours to 15 days), xenografts were removed, and recipients remained alive for up to 287 days. Immunosuppressive therapy based on cyclophosphamide, cyclosporine, and steroids was maintained for 28 days after transplantation. Using appropriate PCR assays, xenochimerism was found in tissue samples and partly even in peripheral blood leukocytes (PBLs) while the porcine kidneys were in situ. After graft removal, xenochimerism was no longer detectable, thus allowing analysis for possible PERV transmission.     

猪内源性逆转录病毒在异种移植中的感染性研究进展

由于人体器官的短缺,猪的器官、组织或细胞成为最可能的供体来源。但猪基因组中整合的内源性逆转录病毒(porcine endogenous retrovirus,PER V)的释放致人体细胞的感染可能性是猪的器官异种移植面临的一大问题。体外研究表明PERV能够感染人类细胞,因此PERV成为异种移植中人们所关心的一个焦点。本文就近年关于PER V在异种移植中的感染性研究进展进行综述。PER V的分子生物学特点及检测方法一、PERV基因组结构Akiyoshi等[1]克隆的猪淋巴细胞PERV的序列长度为8132bp。PER V的基因组结构包括5'端区、型特异性抗原(group鄄specifi…     

Cellular interaction of functional porcine endogenous retrovirus

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.     

No evidence of infection with porcine endogenous retrovirus in recipients of encapsulated porcine islet xenografts

Transplantation of pig tissues into humans has the potential for cotransferring pig infections. Knowledge of the epidemiology of pig infections transmissible to humans allows the development of risk limitation strategies at the source herd level, but potentially infectious pig endogenous retrovirus (PERV) is ubiquitous in all domestic pigs and therefore is not avoidable. Using a specific and sensitive RT-PCR and nested PCR for PERV nucleic acids with primers, the screening of pigs from New Zealand herds for the presence and expression of the PERV was conducted. The presence of PERV proviral DNA (pol and env region) and viral RNA was demonstrated in all tested pig tissues including pancreas, liver, spleen, brain, heart, and PBMC. Using the same assays it was established that different tissues (liver, spleen, and heart) of nude and nonobese diabetic (NOD) mice previously transplanted with nonencapsulated pig islets were PERV DNA and RNA negative. Alginate polylysine capsules prepared with encapsulated pig islets were tested for possible leakage of viral particles or viral nucleic acids. RNA was extracted from the supernatant of viable encapsulated pig islet cells grown in culture for 2 months. No evidence of PERV RNA or of cellular nucleic acids could be found. Two adult type I diabetic subjects were transplanted with 1 x 10(6) neonatal pig islets encased in alginate capsules into the peritoneal cavity. One patient was immunosuppressed. Both showed evidence of graft function (up to 34% reduction in insulin dose, corresponding increase in serum pig C-peptide) for up to 2 years. DNA and RNA were extracted from PBMC and blood plasma of both patients at 19 months posttransplant. No evidence of PERV proviral DNA or RNA could be detected. Piglet islets contain PERV DNA and RNA, but this does not traverse the capsules used or produce any evidence of infection in nude and nonobese diabetic (NOD) mice or humans.     

No transmission of porcine endogenous retrovirus after transplantation of adult porcine islets into diabetic nude mice and immunosuppressed rats

BACKGROUND: The aim of this study was to investigate whether transmission of porcine endogenous retrovirus (PERV) occurs in a model of diabetes reversal by the xenotransplantation of adult porcine islets (APIs) into immunoincompetent diabetic rodents. METHODS: Black-6 nu/nu mice and Lewis rats were immunosuppressed with cyclosporin A (CsA) and FTY 720, and rendered diabetic with streptozotocin. Purified APIs were transplanted into the renal subcapsular space; 5,000 islet equivalents (IEQs) were used in the nude mice (n = 4) and 40,000 IEQs in the rats (n = 4). The nude mice were sacrificed at 75 days after transplantation. In order to confirm chronic xenograft function, the graft-bearing kidney was removed prior to sacrifice. The rats were followed until xenograft rejection, at which time they were sacrificed. Immediately after sacrifice, tissue samples (liver, spleen, and small intestine) were taken for analysis. Quantitative polymerase chain reaction (PCR) was used to assess evidence of PERV transmission, and porcine cell chimerism. RESULTS: All animals became normoglycemic within 48 h of transplantation. The nude mice remained normoglycemic during the 75-day study period, with removal of the graft-bearing kidney resulting in prompt hyperglycemia. The rats remained normoglycemic until xenograft rejection, which occurred at 66 +/- 28 days. Despite the evidence of porcine cell microchimerism in recipients, real-time PCR detected no evidence of PERV transmission in any of the tissue specimens tested. CONCLUSIONS: There was no evidence of PERV transmission following transplantation of pig islets into diabetic nude mice and immunosuppressed rats.     

Development and validation of a Western immunoblot assay for detection of antibodies to porcine endogenous retrovirus

BACKGROUND: Reports that pig endogenous retrovirus (PERV) infects human cells in vitro have heightened the importance of molecular and serologic monitoring of xenograft recipients for evidence of infection with PERV. We report the development and validation of a PERV-specific Western immunoblot assay for the diagnostic testing of porcine xenografts recipients. This assay is based upon the serological cross-reactivity observed between PERV variants capable of infecting human cells in vitro and other mammalian C type retroviruses. METHODS AND RESULTS: Strong reactivity between PERV expressing embryonic pig kidney PK-15 cells and antisera raised against whole virus preparations of murine leukemia virus, gibbon ape leukemia virus (GALV), and simian sarcoma-associated virus was demonstrated by an immunofluorescence assay, suggesting specific antigenic cross-reactivity between this group of viruses and PERV. Western immunoblot analysis demonstrated that anti-GALV antisera reacted with three proteins in PK-15 cells having molecular masses of 30, 55, and 66 kDa. Antisera specific for the Gag proteins of either GALV or simian sarcoma-associated virus reacted with the 30-kDa (major) and 55-kDa (minor) proteins present in PK-15 cells and in PERV-infected 293 human kidney cells, likely representing reactivity to the processed and precursor forms of the PERV Gag protein, respectively. No reactivity was seen in uninfected 293 cells. Analysis of plasma samples from 200 United States blood donors and from 58 human immunodeficiency virus-1, 18 human immunodeficiency virus-2, 13 human T-cell lymphotrophic virus-I, 21 human T-cell lymphotrophic virus-II, and 15 cytomegalovirus infected controls were negative. CONCLUSIONS: As this assay is based on PERV antigen derived from infected human cells, it clearly has the capacity to detect a serologic response towards PERV variants that have zoonotic potential and will allow for the accurate determination of PERV-specific seroreactivity in porcine xenograft recipients.