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

2012 ◽  
Vol 17 (4) ◽  
pp. 92-107 ◽  
Author(s):  
Ewa Nowak
Keyword(s):  
Cyclosporine A ◽  
Endogenous Retroviruses ◽  
Porcine Endogenous Retroviruses

scholarly journals Absence of Replication-Competent Human-Tropic Porcine Endogenous Retroviruses in the Germ Line DNA of Inbred Miniature Swine

2004 ◽  
Vol 78 (5) ◽  
pp. 2502-2509 ◽  
Author(s):  
Linda Scobie ◽  
Samantha Taylor ◽  
James C. Wood ◽  
Kristen M. Suling ◽  
Gary Quinn ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Germ Line ◽  
Human Cells ◽  
Endogenous Retroviruses ◽  
Open Reading Frames ◽  
Miniature Swine ◽  
Dna Libraries ◽  
Porcine Endogenous Retroviruses ◽  
Perv Transmission

ABSTRACT 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.

scholarly journals Establishment and Characterization of Molecular Clones of Porcine Endogenous Retroviruses Replicating on Human Cells

2000 ◽  
Vol 74 (9) ◽  
pp. 4028-4038 ◽  
Author(s):  
Frank Czauderna ◽  
Nicole Fischer ◽  
Klaus Boller ◽  
Reinhard Kurth ◽  
Ralf R. Tönjes
Keyword(s):  
Reverse Transcriptase Activity ◽  
Human Cells ◽  
Endogenous Retroviruses ◽  
Start Codon ◽  
293 Cells ◽  
Porcine Endogenous Retroviruses ◽  
Class B ◽  
Pig Genome ◽  
Genetic Recombinant

ABSTRACT The use of pig xenografts is being considered to alleviate the shortage of allogeneic organs for transplantation. In addition to the problems overcoming immunological and physiological barriers, the existence of numerous porcine microorganisms poses the risk of initiating a xenozoonosis. Recently, different classes of type C porcine endogenous retoviruses (PERV) which are infectious for human cells in vitro have been partially described. We therefore examined whether completely intact proviruses exist that produce infectious and replication-competent virions. Several proviral PERV sequences were cloned and characterized. One molecular PERV class B clone, PERV-B(43), generated infectious particles after transfection into human 293 cells. A second clone, PERV-B(33), which was highly homologous to PERV-B(43), showed a G-to-A mutation in the first start codon (Met to Ile) of the env gene, preventing this provirus from replicating. However, a genetic recombinant, PERV-B(33)/ATG, carrying a restoredenv start codon, became infectious and could be serially passaged on 293 cells similar to virus clone PERV-B(43). PERV protein expression was detected 24 to 48 h posttransfection (p.t.) using cross-reacting antiserum, and reverse transcriptase activity was found at 12 to 14 days p.t. The transcriptional start and stop sites as well as the splice donor and splice acceptor sites of PERV mRNA were mapped, yielding a subgenomic env transcript of 3.1 kb. PERV-B(33) and PERV-B(43) differ in the number of copies of a 39-bp segment in the U3 region of the long terminal repeat. Strategies to identify and to specifically suppress or eliminate those proviruses from the pig genome might help in the production of PERV-free animals.

scholarly journals Characterization of germline porcine endogenous retroviruses from Large White pig

2004 ◽  
Vol 85 (8) ◽  
pp. 2421-2428 ◽  
Author(s):  
Linda Scobie ◽  
Samantha Taylor ◽  
Nicola A. Logan ◽  
Sharon Meikle ◽  
David Onions ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Endogenous Retroviruses ◽  
High Similarity ◽  
Miniature Swine ◽  
Large White ◽  
Safety Aspects ◽  
Porcine Endogenous Retroviruses ◽  
Large White Pig

Porcine endogenous retroviruses (PERV) are of concern when the microbiological safety aspects of xenotransplantation are considered. Four unique isolates of PERV B have been identified previously from a lambda library constructed from genomic DNA from a Large White pig. This study shows that none of these isolates are replication competent when transfected into permissive human or pig cells in vitro, and the removal of flanking genomic sequences does not confer a human tropic replication competent (HTRC) phenotype on these PERV proviruses. Analysis of the envelope sequences revealed that PERV B demonstrated high similarity to the envelope sequences derived from replication-competent PERV, indicating that lack of replication competence does not appear to be attributable to this region of the provirus. These data complement recent findings that HTRC PERV are recombinants between the PERV A and PERV C subgroups, and that these recombinants are not present in the germline of miniature swine. Together, these results indicate that these individual PERV B proviruses are unlikely to give rise to HTRC PERV.

Porcine Endogenous Retroviruses PERV-A and PERV-B Infect neither Mouse Cells in vitro nor SCID Mice in vivo

Intervirology ◽  
2005 ◽  
Vol 48 (2-3) ◽  
pp. 167-173 ◽  
Author(s):  
Markus Irgang ◽  
Alexander Karlas ◽  
Christine Laue ◽  
Volker Specke ◽  
Stefan J. Tacke ◽  
...  
Keyword(s):  
Scid Mice ◽  
Endogenous Retroviruses ◽  
Porcine Endogenous Retroviruses ◽  
Mouse Cells

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 Antiretroviral Agents Inhibit Infection of Human Cells by Porcine Endogenous Retroviruses

2000 ◽  
Vol 44 (12) ◽  
pp. 3432-3433 ◽  
Author(s):  
S. K. Powell ◽  
M. E. Gates ◽  
G. Langford ◽  
M.-L. Gu ◽  
C. Lockey ◽  
...  
Keyword(s):  
Nucleoside Analogs ◽  
Antiretroviral Drugs ◽  
Human Cells ◽  
Endogenous Retroviruses ◽  
Antiretroviral Agents ◽  
Porcine Endogenous Retroviruses

ABSTRACT The efficacy of antiretroviral drugs against porcine endogenous retroviruses (PERV) that may be harbored in pig organs intended for transplantation was examined in human cells in vitro. The nucleoside analogs zidovudine and dideoxyinosine were found to effectively inhibit PERV replication.

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 Extensive Mammalian Germline Genome Engineering

2019 ◽  
Author(s):  
Yanan Yue ◽  
Yinan Kan ◽  
Weihong Xu ◽  
Hong-Ye Zhao ◽  
Yixuan Zhou ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Genome Engineering ◽  
Living Organism ◽  
Endogenous Retroviruses ◽  
Germline Transmission ◽  
Porcine Endogenous Retroviruses ◽  
Pig Genome ◽  
Significant Step ◽  
Porcine Organs

AbstractXenotransplantation, specifically the use of porcine organs for human transplantation, has long been sought after as an alternative for patients suffering from organ failure. However, clinical application of this approach has been impeded by two main hurdles: 1) risk of transmission of porcine endogenous retroviruses (PERVs) and 2) molecular incompatibilities between donor pigs and humans which culminate in rejection of the graft. We previously demonstrated that all 25 copies of the PERV elements in the pig genome could be inactivated and live pigs successfully generated. In this study, we improved the scale of porcine germline editing from targeting a single repetitive locus with CRISPR to engineering 13 different genes using multiple genome engineering methods. we engineered the pig genome at 42 alleles using CRISPR-Cas9 and transposon and produced PERVKO·3KO·9TG pigs which carry PERV inactivation, xeno-antigen KO and 9 effective human transgenes. The engineered pigs exhibit normal physiology, fertility, and germline transmission of the edited alleles. In vitro assays demonstrated that these pigs gain significant resistance to human humoral and cell mediated damage, and coagulation dysregulations, similar to that of allotransplantation. Successful creation of PERVKO·3KO·9TG pigs represents a significant step forward towards safe and effective porcine xenotransplantation, which also represents a synthetic biology accomplishment of engineering novel functions in a living organism.One Sentence SummaryExtensive genome engineering is applied to modify pigs for safe and immune compatible organs for human transplantation

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