scholarly journals Characterization of Porcine Endogenous Retrovirus Clones from the NIH Miniature Pig BAC Library

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Seong-Lan Yu ◽  
Woo-Young Jung ◽  
Kie-Chul Jung ◽  
In-Cheol Cho ◽  
Hyun-Tae Lim ◽  
...  
Keyword(s):  
Bac Library ◽  
Endogenous Retrovirus ◽  
Endogenous Retroviruses ◽  
Miniature Pig ◽  
Reading Frame ◽  
Porcine Endogenous Retrovirus ◽  
Open Reading Frame Length ◽  
Porcine Endogenous Retroviruses ◽  
Integration Sites

Pigs have been considered as donors for xenotransplantation in the replacement of human organs and tissues. However, porcine endogenous retroviruses (PERVs) might transmit new infectious disease to humans during xenotransplantation. To investigate PERV integration sites, 45 PERV-positive BAC clones, including 12 PERV-A, 16 PERV-B, and 17 PERV-C clones, were identified from the NIH miniature pig BAC library. The analysis of 12 selected full-length sequences of PERVs, including the long terminal repeat (LTR) region, identified the expected of open reading frame length, an indicative of active PERV, in all five PERV-C clones and one of the four PERV-B clones. Premature stop codons were observed in only three PERV-A clones. Also, eleven PERV integration sites were mapped using a 5000-rad IMpRH panel. The map locations of PERV-C clones have not been reported before, thus they are novel PERV clones identified in this study. The results could provide basic information for the elimination of site-specific PERVs in selection of pigs for xenotransplantation.

scholarly journals Isolation and Characterization of an Infectious Replication-Competent Molecular Clone of Ecotropic Porcine Endogenous Retrovirus Class C

2006 ◽  
Vol 80 (20) ◽  
pp. 10258-10261 ◽  
Author(s):  
Thomas Preuss ◽  
Nicole Fischer ◽  
Klaus Boller ◽  
Ralf R. Tönjes
Keyword(s):  
T Cells ◽  
Genomic Library ◽  
Endogenous Retrovirus ◽  
Endogenous Retroviruses ◽  
Miniature Swine ◽  
Infectious Risk ◽  
Isolation And Characterization ◽  
Porcine Endogenous Retrovirus ◽  
Porcine Endogenous Retroviruses

ABSTRACT 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.

scholarly journals PCR-based cloning and immunocytological titration of infectious porcine endogenous retrovirus subgroup A and B

2002 ◽  
Vol 83 (9) ◽  
pp. 2231-2240 ◽  
Author(s):  
Birke Bartosch ◽  
Robin A. Weiss ◽  
Yasuhiro Takeuchi
Keyword(s):  
Restriction Site ◽  
Current Method ◽  
Endogenous Retrovirus ◽  
Endogenous Retroviruses ◽  
Porcine Endogenous Retrovirus ◽  
293 Cells ◽  
Cloning Technique ◽  
Unique Restriction ◽  
Human Xenotransplantation

Two pig endogenous retroviruses (PERV), PERV-A and -B, productively infect human cells and are therefore considered to constitute a potential risk in pig-to-human xenotransplantation. A PCR-based cloning technique to isolate infectious PERV proviruses was established. Overlapping 3′ half and 5′ halves of PERV proviral genomes were amplified using DNA extracted from human 293 cells infected with PERV-A or -B. These clones were fused at a unique restriction site in the overlapping region and tested for their infectivity. Representative constructs possessed the same infectious properties as their parent isolates. We also developed a polyclonal anti-PERV serum by using recombinant PERV capsid protein derived from one of the infectious constructs as immunogen and established an immunocytological method for detection and titration of PERV infection. This detection method proved to be more sensitive than the current method of choice (transfer of MLV-lacZ vectors) for infectivity assessment of PERV. These findings should be considered for future characterization of PERV isolates.

scholarly journals Relative Age of Proviral Porcine Endogenous Retrovirus Sequences in Sus scrofa Based on the Molecular Clock Hypothesis

2003 ◽  
Vol 77 (22) ◽  
pp. 12363-12368 ◽  
Author(s):  
Ralf R. Tönjes ◽  
Marcus Niebert
Keyword(s):  
Sus Scrofa ◽  
Endogenous Retrovirus ◽  
Endogenous Retroviruses ◽  
Relative Age ◽  
Repeat Structure ◽  
Porcine Endogenous Retrovirus ◽  
Porcine Endogenous Retroviruses ◽  
Different Types ◽  
Pecari Tajacu ◽  
Recent Origin

ABSTRACT Porcine endogenous retroviruses (PERV) are discussed as putative infectious agents in xenotransplantation. PERV classes A, B, and C harbor different envelope proteins. Two different types of long terminal repeat (LTR) structures exist, of which both are present only in PERV-A. One type of LTR contains a distinct repeat structure in U3, while the other is repeatless, conferring a lower level of transcriptional activity. Since the different LTR structures are distributed unequally among the proviruses and, apparently, PERV is the only virus harboring two different LTR structures, we were interested in determining which LTR is the ancestor. Replication-competent viruses can still be found today, suggesting an evolutionary recent origin. Our studies revealed that the age of PERV is at most 7.6 × 106 years, whereas the repeatless LTR type evolved approximately 3.4 × 106 years ago, being the phylogenetically younger structure. The age determined for PERV correlates with the time of separation between pigs (Suidae, Sus scrofa) and their closest relatives, American-born peccaries (Tayassuidae, Pecari tajacu), 7.4 × 106 years ago.

scholarly journals Genotyping of Porcine Endogenous Retroviruses from a Family of Miniature Swine

2004 ◽  
Vol 78 (1) ◽  
pp. 314-319 ◽  
Author(s):  
Gary Quinn ◽  
James Wood ◽  
Kristen Suling ◽  
Scott Arn ◽  
David H. Sachs ◽  
...  
Keyword(s):  
Endogenous Retrovirus ◽  
Endogenous Retroviruses ◽  
Target Cells ◽  
Nucleotide Polymorphisms ◽  
Miniature Swine ◽  
Porcine Endogenous Retrovirus ◽  
Porcine Endogenous Retroviruses ◽  
The Family ◽  
Transcript Profiles

ABSTRACT The identification of animals in an inbred miniature swine herd that consistently fail to produce replication- competent humantropic porcine endogenous retrovirus (PERV) has prompted studies on the biology of PERV in transmitter and nontransmitter animals. We analyzed PERV RNA transcript profiles in a family of inbred miniature swine (SLAd/d haplotype) in which individual members differed in their capacity to generate humantropic and ecotropic (i.e., pigtropic) virus. We identified unique HaeIII and HpaII gag restriction fragment length polymorphism (RFLP) profiles resulting from single nucleotide polymorphisms in blood cells; these were found only in animals that produced humantropic PERV. These HaeIII and HpaII gag RFLP profiles proved to be components of humantropic PERV as they were transmitted to 293 human target cells in vitro. The humantropic HaeIII and HpaII gag RFLP genotypes in the family of study were not present in other miniature swine in the herd that produced humantropic PERV, indicating that these RFLP profiles relate specifically to this family's lineage.

scholarly journals Genome Areas with High Gene Density and CpG Island Neighborhood Strongly Attract Porcine Endogenous Retrovirus for Integration and Favor the Formation of Hot Spots

2008 ◽  
Vol 83 (4) ◽  
pp. 1920-1929 ◽  
Author(s):  
Y. Moalic ◽  
H. Félix ◽  
Y. Takeuchi ◽  
A. Jestin ◽  
Y. Blanchard
Keyword(s):  
Hot Spots ◽  
Cpg Island ◽  
Hot Spot ◽  
Leukemia Virus ◽  
Cpg Islands ◽  
Endogenous Retrovirus ◽  
Endogenous Retroviruses ◽  
Transcription Start Sites ◽  
Porcine Endogenous Retrovirus ◽  
Integration Sites

ABSTRACT Porcine endogenous retroviruses (PERV) are members of the gammaretrovirus genus and display integration preferences around transcription start sites, a finding which is similar to the preferences of the murine leukemia virus (MLV). Our new genome-wide analysis of the integration profile of a recombinant PERV (PERV A/C), enabled us to examine more than 1,900 integration sites and identify 224 integration hot spots. Investigation of the possible genome features involved in hot-spot formation revealed that the expression level of the genes did not influence distribution of the integration sites of gammaretroviruses (PERV and MLV) or the formation of integration hot spots. However, PERV integration and the presence of hot spots was found to be greater in areas of the genome with high densities of genes with CpG islands. Surprisingly, this was not true for MLV. Simulation of integration profiles revealed that retrovirus integration studies involving the use of the restriction enzyme MseI (widely used in genome integration studies of MLV and gammaretroviral vector) underestimated integration near CpG islands and in gene-dense areas. These results suggest that the integration of gammaretrovirus or gammaretroviral vectors might occur preferentially in genome areas that are highly enriched in genes under CpG island promoter regulation.

scholarly journals Characterization of Chromosomally Assigned Replication-Competent Gamma Porcine Endogenous Retroviruses Derived from a Large White Pig and Expression in Human Cells

2002 ◽  
Vol 76 (6) ◽  
pp. 2714-2720 ◽  
Author(s):  
Marcus Niebert ◽  
Claire Rogel-Gaillard ◽  
Patrick Chardon ◽  
Ralf R. Tönjes
Keyword(s):  
Bac Library ◽  
Artificial Chromosome ◽  
Human Cells ◽  
Endogenous Retroviruses ◽  
Chromosome 1 ◽  
Transplant Proc ◽  
Large White ◽  
Porcine Endogenous Retroviruses

ABSTRACT Vertically transmitted endogenous retroviruses pose an infectious risk in the course of pig-to-human transplantation of cells, tissues, and organs. Two classes of polytropic type C porcine endogenous retroviruses (PERV) productively infect human cells in vitro. The cloning and characterization of replication-competent PERV-B sequences from infected human cells (F. Czauderna, N. Fischer, K. Boller, R. Kurth, and R. R. Tönjes, J. Virol. 74:4028-4038, 2000) as well as the cloning of functional PERV-A and -B sequences from porcine cell line PK15 (U. Krach, N. Fischer, F. Czauderna, and R. R. Tönjes, J. Virol. 75:5465-5472, 2001) have been previously described. Here we report the isolation of four full-length proviral sequences from a porcine bacterial artificial chromosome (BAC) library that comprises chromosomally assigned PERV. Clones Bac-PERV-A(130A12) and Bac-PERV-A(151B10) map to pig chromosome 1 and demonstrate close homology to PK15-PERV-A(58) in env and to PERV-MSL in long terminal repeat (LTR), gag, and pro/pol sequences. Clone Bac-PERV-A(463H12) is located on pig chromosome 3 and demonstrates close homology to PK15-PERV-A(58) in env and to 293-PERV-B(43) in LTR, gag, and pro/pol (Czauderna et al.; R. R. Tönjes, F. Czauderna, N. Fischer, U. Krach, K. Boller, P. Chardon, C. Rogel-Gailard, M. Niebert, G. Scheef, A. Werner, and R. Kurth, Transplant Proc. 32:1158-1161, 2000). Clone Bac-PERV-B(192B9) is located on pig chromosome 7 in the swine leukocyte antigen region and is highly homologous with but distinct from the previously described functional clone 293-PERV-B(43) and bears the number of repeats initially observed in the LTRs of clone 293-PERV-A(42) (Czauderna et al.; Krach et al.). Clones Bac-PERV-A(130A12), Bac-PERV-A(151B10), and Bac-PERV-A(463H12) were replication competent upon transfection into susceptible 293 and HeLa cells. Bac-PERV-B(192B9), however, bears two stop codons in pro/pol preventing this clone from being replication competent in some individual pigs, but initial screenings indicate that this provirus might be intact in others. The data suggest that the porcine genome harbors a limited number of infectious PERV sequences, allowing for specific screening in different pig breeds.

scholarly journals Porcine Endogenous Retrovirus Transmission Characteristics of an Inbred Herd of Miniature Swine

2002 ◽  
Vol 76 (6) ◽  
pp. 3045-3048 ◽  
Author(s):  
Beth A. Oldmixon ◽  
James C. Wood ◽  
Thomas A. Ericsson ◽  
Carolyn A. Wilson ◽  
Mary E. White-Scharf ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Endogenous Retrovirus ◽  
Endogenous Retroviruses ◽  
Receptor Binding Domain ◽  
Transmission Characteristics ◽  
Miniature Swine ◽  
Recombinant Viruses ◽  
Porcine Endogenous Retrovirus ◽  
Porcine Endogenous Retroviruses

ABSTRACT 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.

scholarly journals Identification of a novel type C porcine endogenous retrovirus: evidence that copy number of endogenous retroviruses increases during host inbreeding

2001 ◽  
Vol 82 (8) ◽  
pp. 1829-1834 ◽  
Author(s):  
Rui Mang ◽  
Jolanda Maas ◽  
Xianghong Chen ◽  
Jaap Goudsmit ◽  
Antoinette C. van der Kuyl
Keyword(s):  
Wild Boar ◽  
Copy Number ◽  
Endogenous Retrovirus ◽  
Endogenous Retroviruses ◽  
Human Endogenous Retrovirus ◽  
Porcine Endogenous Retrovirus ◽  
Porcine Endogenous Retroviruses ◽  
Pig Genome ◽  
Multiple Copies ◽  
Type C

Different classes of porcine endogenous retroviruses (PERVs), which have the potential to infect humans during xenotransplantation, have been isolated from the pig genome. Because vertebrate genomes may contain numerous endogenous retrovirus sequences, the pig genome was examined for additional endogenous retroviruses, resulting in the isolation of a novel, complete endogenous retrovirus genome, designated PERV-E. The gag, pol and env genes of PERV-E are closely related to those of human endogenous retrovirus (HERV) 4-1, which belongs to the HERV-E family. Results of studies to determine the presence and copy number of PERVs demonstrated that PERV-E and PERV-A/B-like proviruses were present in all genomes tested, but that PERV-C was not found in two of the species examined, including wild boar. Multiple copies of PERVs could be found in each pig genome. Among all of the pig genomes tested, the wild boar genome had the lowest copy number of all PERVs, suggesting that the number of integrations of complete endogenous retroviruses is increased by inbreeding.

scholarly journals Lack of antibody response in pigs immunized with the transmembrane envelope protein of porcine endogenous retroviruses

2014 ◽  
Vol 95 (8) ◽  
pp. 1827-1831 ◽  
Author(s):  
Martina Keller ◽  
Björn Petersen ◽  
Heiner Niemann ◽  
Joachim Denner
Keyword(s):  
Neutralizing Antibodies ◽  
Mammalian Species ◽  
Neutralization Assay ◽  
Endogenous Retrovirus ◽  
Proximal Region ◽  
Endogenous Retroviruses ◽  
Placental Development ◽  
Porcine Endogenous Retrovirus ◽  
Porcine Endogenous Retroviruses ◽  
Protein P15e

Recently, we immunized different mammalian species (goats, mice, rats, rabbits, guinea pigs and hamsters) with the recombinant ectodomain of the transmembrane envelope (TM) protein p15E of porcine endogenous retrovirus (PERV). In all cases, neutralizing immune sera were induced, which recognized epitopes in the fusion peptide proximal region and the membrane proximal external region of p15E. In order to analyse whether pigs are also able to produce such antibodies, and whether such antibodies can be used to study the involvement of the TM protein in placental development (as was shown for endogenous retroviruses of other species), German landrace pigs were immunized with PERV p15E. No binding and neutralizing antibodies were produced as shown in three Western blot analyses and in a neutralization assay, indicating that pigs are tolerant to their endogenous retroviruses, at least for the ectodomain of the TM protein.

scholarly journals Characterization of Endogenous Retroviruses in Sheep

2003 ◽  
Vol 77 (20) ◽  
pp. 11268-11273 ◽  
Author(s):  
Nikolai Klymiuk ◽  
Mathias Müller ◽  
Gottfried Brem ◽  
Bernhard Aigner
Keyword(s):  
Endogenous Retrovirus ◽  
Ovis Aries ◽  
Endogenous Retroviruses ◽  
Open Reading Frames ◽  
In Vivo Experiments ◽  
Pregnant Sheep ◽  
Reading Frames

ABSTRACT Endogenous retrovirus (ERV) sequences have been found in all mammals. In vitro and in vivo experiments revealed ERV activation and cross-species infection in several species. Sheep (Ovis aries) are used for various biotechnological purposes; however, they have not yet been comprehensively screened for ERV sequences. Therefore, the aim of the study was to classify the ERV sequences in the ovine genome (OERV) by analyzing the retroviral pro-pol sequences. Three OERV β families and nine OERV γ families were revealed. Novel open reading frames (ORF) in the amplified proviral fragment were found in one OERV β family and two OERV γ families. Hybrid OERV produced by putative recombination events were not detected. Quantitative analysis of the OERV sequences in the ovine genome revealed no relevant variations in the endogenous retroviral loads of different breeds. Expression analysis of different tissues from fetal and pregnant sheep detected mRNA from both gammaretrovirus families, showing ORF fragments. Thus, the release of retroviruses from sheep cells cannot be excluded.

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