Absence of replication-competent human-tropic porcine endogenous retroviruses in the germ line DNA of inbred miniature Swine

The potential transmission of porcine endogenous retroviruses (PERVs) has raised concern in the development of porcine xenotransplantation products. Our previous studies have resulted in the identification of animals within a research herd of inbred miniature swine that lack the capacity to transmit PERV to human cells in vitro. In contrast, other animals were capable of PERV transmission. The PERVs that were transmitted to human cells are recombinants between PERV-A and PERV-C in the post-VRA region of the envelope (B. A. Oldmixon, J. C. Wood, T. A. Ericsson, C. A. Wilson, M. E. White-Scharf, G. Andersson, J. L. Greenstein, H. J. Schuurman, and C. Patience, J. Virol. 76:3045-3048, 2002); these viruses we term PERV-A/C. This observation prompted us to determine whether these human-tropic replication-competent (HTRC) PERV-A/C recombinants were present in the genomic DNA of these miniature swine. Genomic DNA libraries were generated from one miniature swine that transmitted HTRC PERV as well as from one miniature swine that did not transmit HTRC PERV. HTRC PERV-A/C proviruses were not identified in the germ line DNAs of these pigs by using genomic mapping. Similarly, although PERV-A loci were identified in both libraries that possessed long env open reading frames, the Env proteins encoded by these loci were nonfunctional according to pseudotype assays. In the absence of a germ line source for HTRC PERV, further studies are warranted to assess the mechanisms by which HTRC PERV can be generated. Once identified, it may prove possible to generate animals with further reduced potential to produce HTRC PERV.     

Isolation and characterization of an infectious replication-competent molecular clone of ecotropic porcine endogenous retrovirus class C

Xenotransplantation of pig organs is complicated by the existence of polytropic replication-competent porcine endogenous retroviruses (PERV) capable of infecting human cells. The potential for recombination between ecotropic PERV-C and human-tropic PERV-A and PERV-B adds another level of infectious risk. Proviral PERV-C were characterized in MAX-T cells derived from d/d haplotype miniature swine. Three proviruses were cloned from a genomic library. Clone PERV-C(1312) generated infectious particles after transfection into porcine ST-IOWA cells. Electron microscopy revealed the same morphologies of virions in MAX-T cells and in ST-IOWA cells infected with cell-free PERV-C(1312) particles, indicating that MAX-T cells harbor one functional PERV-C provirus.     

Identification of residues outside of the receptor binding domain that influence the infectivity and tropism of porcine endogenous retrovirus

Identification of determinants of human tropism of porcine endogenous retrovirus (PERV) is critical to understanding the risk of transmission of PERV to recipients of porcine xenotransplantation products. Previously, we showed that a chimeric envelope cDNA encoding the 360 N-terminal residues of the human-tropic PERV envelope class A (PERV-A) SU and the 130 C-terminal residues of the pig-tropic PERV-C SU and all of TM (PERV-A/C) showed a 100-fold decrease in infectivity titer on human cells (M. Gemeniano, O. Mpanju, D. R. Salomon, M. V. Eiden, and C. A. Wilson, Virology 346:108-117, 2006). To identify residues important for human cell infection, we performed site-directed mutagenesis on each of the nine residues, singly or in combination, that distinguish the C-terminal region of PERV-C from PERV-A. Of the nine amino acids, two single-amino-acid substitutions, Q374R and I412V, restored the infectivity of human cells to the chimeric PERV-A/C to a titer equivalent to that of PERV-A. In contrast, PERV-A/C mutant envelope Q439P resulted in undetectable infection of human cells and an approximately 1,000-fold decrease in control pig cells. Mutation of K441R rescued mutants that carried Q439P, suggesting an incompatibility between the proline residue at this position and the presence of KK in the proteolytic cleavage signal. We confirmed this incompatibility with vectors carrying PERV-A envelope mutant R462K that were also rendered noninfectious. Finally, tropism of vectors carrying PERV-C envelope mutants with only four amino acid changes in the C terminus of PERV-C envelope, NHRQ436YNRP plus K441R, was shifted to one similar to that of PERV-A. Our results show an important and previously unrecognized role for infectivity and tropism for residues at the C terminus of SU.     

Evidence and consequence of porcine endogenous retrovirus recombination

The genetic nature and biological effects of recombination between porcine endogenous retroviruses (PERV) were studied. An infectious molecular clone was generated from a high-titer, human-tropic PERV isolate, PERV-A 14/220 (B. A. Oldmixon, et al. J. Virol. 76:3045-3048, 2002; T. A. Ericsson et al. Proc. Natl. Acad. Sci. USA 100:6759-6764, 2003). To analyze this sequence and 15 available full-length PERV nucleotide sequences, we developed a sequence comparison program, LOHA(TM) to calculate local sequence homology between two sequences. This analysis determined that PERV-A 14/220 arose by homologous recombination of a PERV-C genome replacing an 850-bp region around the pol-env junction with that of a PERV-A sequence. This 850-bp PERV-A sequence encompasses the env receptor binding domain, thereby conferring a wide host range including human cells. In addition, we determined that multiple regions derived from PERV-C are responsible for the increased infectious titer of PERV-A 14/220. Thus, a single recombination event may be a fast and effective way to generate high-titer, potentially harmful PERV. Further, local homology and phylogenetic analyses between 16 full-length sequences revealed evidence for other recombination events in the past that give rise to other PERV genomes that possess the PERV-A, but not the PERV-B, env gene. These results indicate that PERV-A env is more prone to recombination with heterogeneous backbone genomes than PERV-B env. Such recombination events that generate more active PERV-A appear to occur in pigs rather frequently, which increases the potential risk of zoonotic PERV transmission. In this context, pigs lacking non-human-tropic PERV-C would be more suitable as donor animals for clinical xenotransplantation.     

Detailed Mapping of Determinants within the Porcine Endogenous Retrovirus Envelope Surface Unit Identifies Critical Residues for Human Cell Infection within the Proline-Rich Region

Replication-competent porcine endogenous retroviruses (PERVs) are either human cell tropic (PERV-A and PERV-B) or non-human cell tropic (PERV-C). We previously demonstrated that PERV in vitro cell tropism is modulated by 2 residues within the C terminus of SU and that the PERV receptor binding domain (RBD) extends beyond the variable regions A and B (VRA and VRB, respectively), to include the proline rich-region (PRR) of SU (M. Gemeniano et al., Virology 346:108-117, 2000; T. Argaw et al., J. Virol. 82:7483-7489, 2008). The present study aimed to identify the specific elements within the PERV RBD that interact with the C-terminal elements of SU to facilitate human cell infection. We constructed a series of chimeric and mutated envelopes between PERV-A and PERV-C and using pseudotyped retroviral vectors to map the human cell tropism-determining sequences within the PERV RBD. We show that the PRR from PERV-A is both necessary and sufficient to allow human cell infection when substituted into the homologous region of the PERV-C envelope carrying two C-terminal amino acid substitutions shown to influence human cell tropism, Q374R and I412V (PERV-Crv). Furthermore, substitution of a single amino acid residue in the PRR of the non-human-tropic PERV-Crv envelope allows vectors carrying this envelope to infect human cells. Receptor interference assays showed that these modified PERV-C envelopes do not bind either of the human PERV-A receptors, suggesting the presence of a distinct human PERV-C receptor. Finally, vectors carrying these modified PERV-C envelopes infect primary human endothelial cells, a cell type likely to be exposed to PERV in clinical use of certain porcine xenotransplantation products.     

Determinants of high titer in recombinant porcine endogenous retroviruses

Porcine endogenous retroviruses (PERVs) pose a potential stumbling block for therapeutic xenotransplantation, with the greatest threat coming from viruses generated by recombination between members of the PERV subgroup A (PERV-A) and PERV-C families (PERV-A/C recombinants). PERV-A and PERV-B have been shown to infect human cells in culture, albeit with low titers. PERV-C has a more restricted host range and cannot infect human cells. A recombinant PERV-A/C virus (PERV-A14/220) contains the PERV-A sequence between the end of pol and the middle of the SU region in env. The remaining sequence is derived from PERV-C. PERV-A14/220 is approximately 500-fold more infectious than PERV-A. To determine the molecular basis for the increased infectivity of PERV-A14/220, we have made a series of vector constructs. The primary determinant for the enhanced replicative potential of the recombinant virus appeared to be the env gene. Using a series of chimeric env genes, we could identify two determinants of high infectivity; one was an isoleucine to valine substitution at position 140 between variable regions A and B, and the other lies within the proline rich region. Taken together, these results show that the novel juxtaposition of env gene sequences enhanced the infectivity of PERV-A14/220 for human cells, perhaps by stabilization of the envelope glycoprotein or increased receptor binding.     

Absence of replication of porcine endogenous retrovirus and porcine lymphotropic herpesvirus type 1 with prolonged pig cell microchimerism after pig-to-baboon xenotransplantation

Porcine endogenous retrovirus (PERV), porcine cytomegalovirus (PCMV), and porcine lymphotropic herpesvirus (PLHV) are common porcine viruses that may be activated with immunosuppression for xenotransplantation. Studies of viral replication or transmission are possible due to prolonged survival of xenografts in baboon recipients from human decay-accelerating factor transgenic or alpha-1,3-galactosyltransferase gene knockout miniature swine. Ten baboons underwent xenotransplantation with transgenic pig organs. Graft survival was 32 to 179 days. Recipient serial samples of peripheral blood mononuclear cells (PBMC) and plasma were analyzed for PCMV, PERV, and PLHV-1 nucleic acids and viral replication using quantitative PCR assays. The PBMC contained PERV proviral DNA in 10 animals, PLHV-1 DNA in 6, and PCMV in 2. PERV RNA was not detected in any PBMC or serum samples. Plasma PLHV-1 DNA was detected in one animal. Pig cell microchimerism (pig major histocompatibility complex class I and pig mitochondrial cytochrome c oxidase subunit II sequences) was present in all recipients with detectable PERV or PLHV-1 (85.5%). Productive infection of PERV or PLHV-1 could not be demonstrated. The PLHV-1 viral load did not increase in serum over time, despite prolonged graft survival and pig cell microchimerism. There was no association of viral loads with the nature of exogenous immune suppression. In conclusion, PERV provirus and PLHV-1 DNA were detected in baboons following porcine xenotransplantation. Viral detection appeared to be due to persistent pig cell microchimerism. There was no evidence of productive infection in recipient baboons for up to 6 months of xenograft function.     

Porcine endogenous retrovirus transmission characteristics of an inbred herd of miniature swine

Here we report the identification of inbred miniature swine that failed to produce human-tropic replication-competent porcine endogenous retroviruses (HTRC PERVs), using in vitro coculture assays. When HTRC PERVs were isolated from transmitting animals, all were recombinant viruses, with the receptor-binding domain of PERV-A combining with PERV-C-related sequences.     

Analysis of natural recombination in porcine endogenous retrovirus envelope genes

Human tropic Porcine Endogenous Retroviruses (PERVs) are the major concern in zoonosis for xenotransplantation because PERVs cannot be eliminated by specific pathogen-free breeding. Recently, a PERV A/C recombinant with PERV-C bearing PERV-A gp70 showed a higher infectivity (approximately 500-fold) to human cells than PERV-A. Additionally, the chance of recombination between PERVs and HERVs is frequently stated as another risk of xenografting. Overcoming zoonotic barriers in xenotransplantation is more complicated by recombination. To achieve successful xenotransplantation, studies on the recombination in PERVs are important. Here, we cloned and sequenced proviral PERV env sequences from pig gDNAs to analyze natural recombination. The envelope is the most important element in retroviruses as a pivotal determinant of host tropisms. As a result, a total of 164 PERV envelope genes were cloned from pigs (four conventional pigs and two miniature pigs). Distribution analysis and recombination analysis of PERVs were performed. Among them, five A/B recombinant clones were identified. Based on our analysis, we determined the minimum natural recombination frequency among PERVs to be 3%. Although a functional recombinant envelope clone was not found, our data evidently show that the recombination event among PERVs may occur naturally in pigs with a rather high possibility.     

猪内源性反转录病毒在中国实验小型猪中的存在与表达

目的对中国实验小型猪中内源性反转录病毒的存在与mRNA的表达进行检测,摸清中国实验小型猪中内源性反转录病毒的携带情况.方法根据已发表的PERV的序列设计并合成了三对引物,分别用于检测PERV核心蛋白基因(gag)、多聚酶基因(pol)及囊膜基因(env)的存在与表达;同时,根据目前通用的env基因分型方法合成了三对用于分型检测的引物env-A、env-B、env-C.应用PCR、RT-PCR扩增的方法,对来自于中国实验小型猪外周血淋巴细胞的DNA和RNA样品进行了检测.结果在6个被检DNA样品中均检出了PERV特异性DNA的存在;同样,在被检RNA样品中均有PERV特异性RNA的表达,且所表达的PERV均为A型和B型,在所有样品中均未检出C型PERV的表达.结论初步表明中国实验小型猪中存在内源性反转录病毒序列,且能以mRNA的形式表达,这一结果为我国特有小型猪的开发、利用及其病毒安全性评价奠定了基础.     

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.     

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.     

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。     

Characterization of PERV in a new conserved pig herd as potential donor animals for xenotransplantation in China

Background

Xenotransplantation has drawn increased attention in recent years as a potential solution to the scarcity of human source donor organs. Researchers have highlighted the need to characterize the influence of porcine endogenous retroviruses (PERV) in xenotransplantation. Screening and analyzing the presence and subtype of PERV in donor source animal breeds could provide basic parameters to evaluate the biological safety of xenotransplantation from pigs to humans. We bred a new miniature porcine herd (XENO-1) after decades of investigation, the herd was purpose bred to produce a potential donor animal source for xenotransplantation. To this end we studied the animals’ PERV expression characteristics.

Methods

We randomly selected 37 animals of the herd, PCR and RT-PCR based on specific primers were utilized to determine their PERV viral subtype. High fidelity PCR and restriction enzyme digestion were employed for variants detection. To thoroughly understand the PERV expression pattern, quantitative PCR was applied to measure mRNA expression levels in different tissues, At last, transfection capacity was assessed using a in vitro co-culture system.

Results

Our results revealed that the XENO-1 herd was free of PERV-C and exhibited low levels of PERVs in different tissues compared to commercial pig (landrace). The XENO-1 herd showed unique variants of A/B recombination. In addition, even though there were A/B variants in the XENO-1 herd, co-culturing revealed no evidence of PERV transmission from XENO-1 tissue to human cells.

Conclusion

Overall, Our results displayed an unique PERV expression pattern in a new pig herd and demonstrated its non-transfection capacity in vitro. Data in the research indicate that XENO-1 animals can serve as a better potential donor source for xenotransplantation.

     

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.     

Multiple groups of novel retroviral genomes in pigs and related species

In view of the concern over potential infection hazards in the use of porcine tissues and organs for xenotransplantation to humans, we investigated the diversity of porcine endogenous retrovirus (PERV) genomes in the DNA of domestic pigs and related species. In addition to the three known envelope subgroups of infectious gamma retroviruses (PERV-A, -B, and -C), classed together here as PERV group gamma 1, four novel groups of gamma retrovirus (gamma 2 to gamma 5) and four novel groups of beta retrovirus (beta 1 to beta 4) genomes were detected in pig DNA using generic and specific PCR primers. PCR quantification indicated that the retroviral genome copy number in the Landrace x Duroc F(1) hybrid pig ranged from 2 (beta 2 and gamma 5) to approximately 50 (gamma 1). The gamma 1, gamma 2, and beta 4 genomes were transcribed into RNA in adult kidney tissue. Apart from gamma 1, the retroviral genomes are not known to be infectious, and sequencing of a small number of amplified genome fragments revealed stop codons in putative open reading frames in several cases. Analysis of DNA from wild boar and other species of Old World pigs (Suidae) and New World peccaries (Tayassuidae) showed that one retrovirus group, beta 2, was common to all species tested, while the others were present among all Old World species but absent from New World species. The PERV-C subgroup of gamma1 genomes segregated among domestic pigs and were absent from two African species (red river hog and warthog). Thus domestic swine and their phylogenetic relatives harbor multiple groups of hitherto undescribed PERV genomes.