Cloning and characterization of replication-competent ecotropic porcine endogenous retroviruses (PERV-C) in the genome of pigs used and intended for clinical pig-to-human xenotransplantation

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.

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

Journal of Virology ◽

10.1128/jvi.01140-06 ◽

2006 ◽

Vol 80 (20) ◽

pp. 10258-10261 ◽

Cited By ~ 17

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.

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

Journal of General Virology ◽

10.1099/0022-1317-83-9-2231 ◽

2002 ◽

Vol 83 (9) ◽

pp. 2231-2240 ◽

Cited By ~ 30

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.

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

Journal of Biomedicine and Biotechnology ◽

10.1155/2012/482568 ◽

2012 ◽

Vol 2012 ◽

pp. 1-10 ◽

Cited By ~ 4

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.

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

Journal of Virology ◽

10.1128/jvi.76.6.2714-2720.2002 ◽

2002 ◽

Vol 76 (6) ◽

pp. 2714-2720 ◽

Cited By ~ 47

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.

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Unexpected low expression of porcine endogenous retroviruses (PERVs) in porcine expanded potential stem cells (EPSCs)

Virus Research ◽

10.1016/j.virusres.2021.198295 ◽

2021 ◽

pp. 198295

Author(s):

Luise Krüger ◽

Monika Nowak-Imialek ◽

Yannick Kristiansen ◽

Doris Herrmann ◽

Björn Petersen ◽

...

Keyword(s):

Stem Cells ◽

Endogenous Retroviruses ◽

Porcine Endogenous Retroviruses ◽

Low Expression

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

Journal of Virology ◽

10.1128/jvi.78.5.2502-2509.2004 ◽

2004 ◽

Vol 78 (5) ◽

pp. 2502-2509 ◽

Cited By ~ 46

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.

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