Activation of cytomegalovirus in pig-to-primate organ xenotransplantation

Xenotransplantation of porcine organs carries the risk of reactivation of latent virus in donor and recipient tissues as well as transmission of viruses between species. We have investigated the activation of baboon cytomegalovirus (BCMV) and porcine CMV (PCMV) in a pig-to-primate model of xenotransplantation. Tissues originating from a series of six swine-to-baboon composite thymokidney xenotransplants were investigated. Four immunosuppressed baboons died (survival range, 7 to 27 days) with the graft in situ. Increases in BCMV DNA copy numbers occurred in three (75%) of these baboons and was thought to be responsible for pneumonitis and the death of one animal. In two baboons, disseminated intravascular coagulation was successfully treated by graftectomy and discontinuation of immunosuppression. PCMV was upregulated in five of six xenografts (83%). PCMV infection was associated with ureteric necrosis in one xenograft. Although significantly increased in native tissues, low levels of BCMV and PCMV were also detected in tissues other than that of the native viral host species. The cross-species presence of CMV did not appear to cause clinical or histological signs of invasive disease. Thus, viral infections with clinical disease were restricted to tissues of the native species of each virus. Intensive immune suppression currently required for xenotransplantation results in a significant risk of reactivation of latent infections by BCMV and PCMV. It is not yet known whether viral DNA detected across species lines represents cellular microchimerism, ongoing viral infection, or uptake of free virus. The observation of graft injury by PCMV demonstrates that CMV will be an important pathogen in immunosuppressed xenograft recipients. Strategies must be developed to exclude CMV from porcine organ donors.     

猪皮肤成纤维细胞PERV体外和体内感染性的研究

为了解猪皮肤成纤维细胞PERV在体外和体内的感染性,通过建立猪皮肤成纤维细胞系,将所建细胞系与人胚胎肾293细胞体外共培养,并移植于严重联合免疫缺陷鼠(SCID鼠)皮下进行猪皮肤成纤维细胞PERV的体外和体内感染性实验。结果表明,猪皮肤成纤维细胞与人胚胎肾细胞共培养过程中,猪内源性逆转录病毒感染人胚胎肾细胞,进一步证实和拓宽了猪细胞PERV感染人细胞的范畴;猪皮肤成纤维细胞移植SCID鼠皮下后,导致SCID鼠发生猪细胞微嵌合(78．57％)和PERV在体内感染(85．71％)并且波及远离移植部位的多种组织或器官,但是并未检测出SCID鼠组织中表达PERV env RNA。这就证实了猪皮肤成纤维细胞PERV的体外感染性和在小鼠体内的感染性,但未能找到PERV在体内活跃复制的明显证据。因而,在猪异种移植过程中PERV传播的潜在危险仍然是必须高度重视的生物安全性问题。     

Mice transgenic for a human porcine endogenous retrovirus receptor are susceptible to productive viral infection

Porcine endogenous retrovirus (PERV) is considered one of the major risks in xenotransplantation. No valid animal model has been established to evaluate the risks associated with PERV transmission to human patients by pig tissue xenotransplantation or to study the potential pathogenesis associated with PERV infection. In previous work we isolated two genes encoding functional human PERV receptors and proved that introduction of these into mouse fibroblasts allowed the normally nonpermissive mouse cells to become productively infected (T. A. Ericsson, Y. Takeuchi, C. Templin, G. Quinn, S. F. Farhadian, J. C. Wood, B. A. Oldmixon, K. M. Suling, J. K. Ishii, Y. Kitagawa, T. Miyazawa, D. R. Salomon, R. A. Weiss, and C. Patience, Proc. Natl. Acad. Sci. USA 100:6759-6764, 2003). In the present study we created mice transgenic for human PERV-A receptor 2 (HuPAR-2). After inoculation of transgenic animals with infectious PERV supernatants, viral DNA and RNA were detected at multiple time points, indicating productive replication. This establishes the role of HuPAR-2 in PERV infection in vivo; in addition, these transgenic mice represent a new model for determining the risk of PERV transmission and potential pathogenesis. These mice also create a unique opportunity to study the immune response to PERV infection and test potential therapeutic or preventative modalities.     

Identification of exogenous forms of human-tropic porcine endogenous retrovirus in miniature Swine

The replication of porcine endogenous retrovirus subgroup A (PERV-A) and PERV-B in certain human cell lines indicates that PERV may pose an infectious risk in clinical xenotransplantation. We have previously reported that human-tropic PERVs isolated from infected human cells following cocultivation with miniature swine peripheral blood mononuclear cells (PBMC) are recombinants of PERV-A with PERV-C. Here, we report that these recombinants are exogenous viruses in miniature swine; i.e., they are not present in the germ line DNA. These viruses were invariably present in miniature swine that transmitted PERV to human cells and were also identified in some miniature swine that lacked this ability. These data, together with the demonstration of the absence of both replication-competent PERV-A and recombinant PERV-A/C loci in the genome of miniature swine (L. Scobie, S. Taylor, J. C. Wood, K. M. Suling, G. Quinn, C. Patience, H.-J. Schuurman, and D. E. Onions, J. Virol. 78:2502-2509, 2004), indicate that exogenous PERV is the principal source of human-tropic virus in these animals. Interestingly, strong expression of PERV-C in PBMC correlated with an ability of the PBMC to transmit PERV-A/C recombinants in vitro, indicating that PERV-C may be an important factor affecting the production of human-tropic PERV. In light of these observations, the safety of clinical xenotransplantation from miniature swine will be most enhanced by the utilization of source animals that do not transmit PERV to either human or porcine cells. Such animals were identified within the miniature swine herd and may further enhance the safety of clinical xenotransplantation.     

Inhibition of budding/release of porcine endogenous retrovirus

PERV is integrated into the genome of all pigs. PERV‐A and PERV‐B are polytropic and can productively infect human cell lines, whereas PERV‐C is ecotropic. Recombinant PERV‐A/C can infect human cells and exhibits high titer replication. Therefore, use of pigs for human xenotransplantation raises concerns about the risks of transfer of this infectious agent from donors to xenotransplantation recipients. To establish strategies to inhibit PERV production from cells, in the present study, we investigated the mechanism of PERV budding and anti‐PERV activity of Tetherin/BST‐2. The results showed that DN mutants of WWP‐2, Tsg101, and Vps4A/B markedly reduced PERV production in human and porcine cell lines, suggesting that PERV budding uses these cellular factors and the cellular MVB sorting pathway as well as many other retroviruses. Moreover, PERV production was also reduced by human and porcine Tetherin/BST‐2. These data are useful for developing strategies to inhibit PERV production and may reduce the risk of PERV infection in xenotransplantation.     

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.     

湖南沙子岭猪内源性逆转录病毒的研究

为评价从猪到人异种移植的生物安全性提供依据,从湖南沙子岭猪的保种群内随机采集31头个体的耳样组织,应用PCR和RT-PCR技术分别检测这些组织中内源性逆转录病毒（porcine endogenous retrovirus,PERV）的前病毒DNA和mRNA,并对PCR扩增的灵敏性进行评估。多组织RT-PCR检测3头沙子岭猪肾、心、肝、肺、脾 等组织中PERV的表达情况,了解其在各组织中的分布情况;最后,扩增、测序该猪种的env基因,结果用NCBI中的BLAST软件进行分析。PCR和RT-PCR结果表明,所检测的31头沙子岭猪均带有PERV前病毒DNA,耳样组织中均有PERV mRNA表达,其中有2头个体携带 env-A、env-B、env-C 3种囊膜蛋白基因,而其余的29头个体只带有env-A、env-B 两种囊膜蛋白基因,未检测到env-C基因。多组织RT-PCR扩增结果表明,3头沙子岭猪的肾、心、肝、肺、脾等组织中,pol、gag、env-A、env-B 基因均有表达,未检测到env-C基因表达。测序沙子岭猪的env基因,结果发现,沙子岭猪env-B 和env-C基因与其他猪种序列比较分别存在2 和10个碱基的差异,而env-A基因序列没有差异,说明不同的猪种之间 env基因存在多态性。以上结果表明,沙子岭猪种群携带PERV,其亚型主要以PERV-A,B为主;PERV在该猪种肾、心、肝、肺、脾等多种组织中的分布没有明显组织特异性,且93.5 % (29/31)个体表现为 env-C 基因缺失,提示沙子岭猪作为候选猪种可能在异种移植中具有较好的应用前景。     

Infection of nonhuman primate cells by pig endogenous retrovirus

The ongoing shortage of human donor organs for transplantation has catalyzed new interest in the application of pig organs (xenotransplantation). One of the biggest concerns about the transplantation of porcine grafts into humans is the transmission of pig endogenous retroviruses (PERV) to the recipients or even to other members of the community. Although nonhuman primate models are excellently suited to mimic clinical xenotransplantation settings, their value for risk assessment of PERV transmission at xenotransplantation is questionable since all of the primate cell lines tested so far have been found to be nonpermissive for PERV infection. Here we demonstrate that human, gorilla, and Papio hamadryas primary skin fibroblasts and also baboon B-cell lines are permissive for PERV infection. This suggests that a reevaluation of the suitability of the baboon model for risk assessment in xenotransplantation is critical at this point.     

Restriction of porcine endogenous retrovirus by porcine APOBEC3 cytidine deaminases

Xenotransplantation of porcine cells, tissues, and organs shows promise to surmount the shortage of human donor materials. Among the barriers to pig-to-human xenotransplantation are porcine endogenous retroviruses (PERV) since functional representatives of the two polytropic classes, PERV-A and PERV-B, are able to infect human embryonic kidney cells in vitro, suggesting that a xenozoonosis in vivo could occur. To assess the capacity of human and porcine cells to counteract PERV infections, we analyzed human and porcine APOBEC3 (A3) proteins. This multigene family of cytidine deaminases contributes to the cellular intrinsic immunity and act as potent inhibitors of retroviruses and retrotransposons. Our data show that the porcine A3 gene locus on chromosome 5 consists of the two single-domain genes A3Z2 and A3Z3. The evolutionary relationships of the A3Z3 genes reflect the evolutionary history of mammals. The two A3 genes encode at least four different mRNAs: A3Z2, A3Z3, A3Z2-Z3, and A3Z2-Z3 splice variant A (SVA). Porcine and human A3s have been tested toward their antiretroviral activity against PERV and murine leukemia virus (MuLV) using novel single-round reporter viruses. The porcine A3Z2, A3Z3 and A3Z2-Z3 were packaged into PERV particles and inhibited PERV replication in a dose-dependent manner. The antiretroviral effect correlated with editing by the porcine A3s with a trinucleotide preference for 5' TGC for A3Z2 and A3Z2-Z3 and 5' CAC for A3Z3. These results strongly imply that human and porcine A3s could inhibit PERV replication in vivo, thereby reducing the risk of infection of human cells by PERV in the context of pig-to-human xenotransplantation.     

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.     

Suboptimal porcine endogenous retrovirus infection in non-human primate cells: implication for preclinical xenotransplantation

Background

Porcine 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 Findings

Two 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.

Conclusions

Since 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.     

PERV在猪外周血白细胞DNA和组织mRNA中的表达及其差异性分析

为了解我国家猪猪内源性逆转录病毒(PERV)生物学的基本特征,为评价应用猪器官、组织、细胞进行猪一人间跨种移植的生物安全性提供理论基础。本文采用PCR方法调查12个家猪品系外周血白细胞DNA基因组PERV的生物学特征,并应用SS-SSCP、RFLP-PCR方法分析PERV基因片段的差异性及采用RT-PCR方法和半定量方法分析2个品系小型猪13种组织PERV表达的差异。结果表明12个品系猪外周血白细胞DNA基因组普遍存在PERV-A、-B基因序列,未发现单链构象多态性;部分品系猪PER Venv基因序列片段存在限制性片段长度多态性。分析2个品系13种组织均表达PERV-A、-B、-C,肾、淋巴结、肝为高表达器官,胰腺和脑组织为低表达器官,PERV-C mRNA丰度明显低于PERV-A、-B mRNA。PERV env存在限制性片段长度多态性、PERV-A存在碱基缺失和错配的现象,有可能在猪异种移植中构成PERV感染的潜在危险性,这是在猪异种移植过程中值得高度关注的问题。     

Rapid determination of perv copy number from porcine genomic DNA by real-time polymerase chain reaction

Here, we report the quantification of porcine endogenous retrovirus (PERV) copy numbers using real time PCR. After generating standard curves using plasmid DNA, copy numbers were determined for PERV pol and for a housekeeping gene, porcine estrogen receptor2 (ER2) with the same amount of genomic DNA. Using this method, we examined 6 pig breeds in Korea including two breeds of miniature pig, one domestic pig from Jeju, and imported pig breeds, Duroc, Landrace, and Yorkshire. All breeds showed PERV copy numbers ranging from 9 to 50. This method will be useful for monitoring of PERVs in a porcine xenograft.