Extended Analysis of the In Vitro Tropism of Porcine Endogenous Retrovirus

Wilson, Carolyn A.; Wong, Susan; VanBrocklin, Matthew W.; Federspiel, Mark J.

doi:10.1128/jvi.74.1.49-56.2000

Cited by 197 publications

(244 citation statements)

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“…The ability of PERV produced by pig cell lines or primary cell cultures to infect human cells in vitro has been documented [8,9,13,14,15,16], and serial passages of PERV on a human cell line have produced retroviruses with a higher tropism for human cells [17]. Our previous work found that pancreas expressed the lowest PERV mRNA levels in SPF pig tissues intended for grafting [5] and that long-term follow-up failed to detect in vitro transmission of PERV from SPF pig islets to a panel of human cells (including very permissive 293 tumour cells), even after repeated and optimised co-incubations [8].…”

Section: Discussionmentioning

confidence: 99%

“…However, there is also a risk of transmitting porcine endogenous retroviruses (PERV) [9,10,11,12]. The ability of PERV [10] produced by pig cells to infect human cells in vitro has been documented [8,9,13,14,15,16,17]. PERV mRNA and full-length endogenous retroviral genome were expressed in all pigs tested [9,15,18,19], including SPF pigs [5,20].…”

Section: Pcr-derived Monitoring Of Perv Infection and Microchimerism mentioning

confidence: 99%

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Microchimerism and transmission of porcine endogenous retrovirus from a pig cell line or specific pathogen-free pig islets to mouse tissues and human cells during xenografts in nude mice

et al. 2002

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Aims/hypothesis. Pig islets could transmit porcine endogenous retroviruses (PERV) to diabetic patients. Our previous work showed that pig islets expressed low levels of PERV mRNA and were not likely to transmit PERV to human cells in vitro. The real risk of infection during pig tissue xenografts can only be evaluated by in vivo experiments. Methods. Nude mice bearing tumours containing human 293 cells were grafted with specific pathogenfree pig islets or PERV-producing pig PK15 cells to determine whether pig cells could transmit PERV to mouse and human cells in vivo. Infection was monitored by PCR, long PCR, RT-PCR and long RT-PCR. As detection of PERV sequences could be due to the presence of residual pig cells, we looked for pig mitochondrial (mt) DNA. Quantitative PCR for PERV and pig mt DNA was done to compare the PERV-topig mt (P-to-M) ratio of each sample with the reference ratio for grafted pig cells. Results. Among 78 mouse tissues from PK15-grafted mice, 54 and 72 were positive for gag and pig mt DNA, respectively. Human tumours developed in these mice were positive for PERV (78%) and pig mt (89%). The P-to-M ratios for mouse tissues and PERV-positive human tumours from PK15-grafted mice were higher than the ratio in PK15 cells. Among 41 tissues from pig islet cell-grafted mice, 7 were positive for PERV (3 lymph nodes, 1 kidney, 2 salivary glands, 1 ovary), and 14 were positive for pig mt DNA. Three of these samples (1 lymph node, 1 kidney and 1 salivary gland) were positive for gag DNA, but negative for pig mt DNA. One human tumour in these mice was positive for PERV DNA. P-to-M reference ratio in grafted islet cells was 0.05±0.03. The three PERVpositive lymph nodes contained 78 gag/3 mt copies (P-to-M: 26), 101 gag/3 mt copies (P-to-M: 34), and 4 gag/0 mt copies. The two PERV-positive salivary glands contained 14 gag/1 mt copies, and 28 gag/0 mt copies. The ovary and the kidney contained 46 gag/ 3 mt and 69 gag/0 mt copies, respectively. The PERV-positive human tumour contained 47 gag/3 mt copies. Conclusions/interpretation. Microchimerism and PERV transmission were frequently observed in both mouse and human tissues during grafting of pig PK15 cells into nude mice bearing human tumours, and sometimes during pig islet xenograft in this model. This strengthens the notion that there is a risk of transmitting PERV during pig islet xenograft. [Diabetologia (2002) 45:914-923] Keywords Xenograft, pig endogenous retrovirus, pig islet cell, PK15 cells, specific pathogen free pig, human cells, mouse. vided by E. Gouin (Ploufragan and Nantes, France) from Large-White SPF pigs (80-120 kg), aged 20 weeks, as previously described [1,2]. Pancreases were removed in betadine solution and inflated with 100 ml ice-cold sterile modified University of Wisconsin (mUW) solution and transported in mUW. Pancreases were then inflated again with 0.5 ml/g of mUW solution containing 2 mg/ml Liberase (Roche Diagnostics, Meylan, France) and placed in a digestion chamber filled with mUW solution kept at 36°C. Crude isle...

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Section: Discussionmentioning

confidence: 99%

Section: Pcr-derived Monitoring Of Perv Infection and Microchimerism mentioning

confidence: 99%

Microchimerism and transmission of porcine endogenous retrovirus from a pig cell line or specific pathogen-free pig islets to mouse tissues and human cells during xenografts in nude mice

et al. 2002

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“…Other molecular assays, such as generic detection of RT in plasma or culture recipient cells using ultra sensitive PCR-based RT assays, may be helpful in the detection of novel or variant pig retroviruses that may be transmitted from porcine xenografts [13][14][15].…”

Section: Discussionmentioning

confidence: 99%

Polymerase chain reaction in detection of porcine endogenous retrovirus (PERV) from porcine tissues

Prabha

Verghese

2009

Indian J Microbiol

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Pigs offer an unlimited source of xenografts for humans. The use of transplants from animal origin offers a potential solution to the limited supply of human organs and tissues. However, like many other mammalian species, pigs harbor porcine endogenous retrovirus (PERV), which are encoded in their genomic DNA and are assumed to have been integrated into the porcine germline. The ability of PERV to infect human cells in vitro has heightened safety concerns regarding the transmission of PERV to pig xenograft recipients. Porcine tissues were analyzed using validated assays specifi c for PERV: polymerase chain reaction (PCR) (for PERV DNA) and reverse transcriptase (RT)-PCR (for PERV RNA). PERV-specifi c gag sequences were found in the porcine heart tissue samples using DNA-PCR and RT-PCR. PCR is a rapid and specifi c test for the detection of PERV from xenografts. These fi ndings have demonstrated that the presence of both DNA and RNA forms of PERV in porcine tissues needs to be carefully considered when the infectious disease potential of xenotransplantation is being assessed.

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“…Peptide II (EIATL*PLRT), representing the MA*CA site of PERV-C, had an eightfold-higher K m , implying lower cleavage and probably maturation rates of the subtype C virus. Interesting, in this context, may be the fact that PERV C has been shown to have replication disadvantages in human cells (18,19).…”

mentioning

confidence: 99%

Molecular and Enzymatic Characterization of the Porcine Endogenous Retrovirus Protease

Blusch

Seelmeir

Helm

2002

J Virol

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The protease of the porcine endogenous retrovirus (PERV) subtypes A/B and C was recombinantly expressed in Escherichia coli as proteolytically active enzyme and characterized. The PERV Gag precursor was also recombinantly produced and used as the substrate in an in vitro enzyme assay in parallel with synthetic nonapeptide substrates designed according to cleavage site sequences identified in the PERV Gag precursor. The proteases of all PERV subtypes consist of 127 amino acid residues with an M r of 14,000 as revealed by determining the protease N and C termini. The PERV proteases have a high specificity for PERV substrates and do not cleave human immunodeficiency virus (HIV)-specific substrates, nor are they inhibited by specific HIV protease inhibitors. Among the known retroviral proteases, the PERV proteases resemble most closely the protease of the murine leukemia retrovirus.Organ xenotransplantation from pigs may be one possibility to solve the shortage of human organ donors. An unanswered question is, however, the risk of viral infection by porcine endogenous retroviruses (PERVs) upon xenografting (3,5,7,10,11,16). One way to minimize this problem might be the option of designing an effective antiviral chemotherapy against PERV, comparable to that presently employed against human immunodeficiency virus (HIV) infection. Here we have characterized the PERV protease as a target for protease inhibitors (PIs) that might be employed as antiviral agents for chemotherapy.Three main subtypes of PERV, A, B, and C, are known to date (14). All are classical C-type retroviruses (e.g., 1, 2, 15) and are genetically similar but not identical. We have revealed the coding sequences for the proteases of all three PERV subtypes, produced active proteases by recombinant methods in Escherichia coli, and established an in vitro assay system for PERV protease activity.Coding sequence of the PERV protease. For recombinant expression of the PERV protease, the boundaries of its coding sequence had to be revealed. Possible N and C termini of the functional PERV protease coding sequence were predicted by multiple alignment of the translated sequences of gag and pol regions of PERV subtypes A/B (DuxDL3791 [J. Blusch et al., unpublished data]) and C (PERV-MSL [1]) with the protease sequence of Moloney murine leukemia virus (MMLV [8,20]), the phylogenetically closest C-type retrovirus with a known protease protein sequence (Fig. 1).The alignment indicated that the C terminus of the PERV protease seemed to be highly similar to that of the murine leukemia virus protease; the N terminus of PERV protease, however, showed significant differences (Fig. 1). The N terminus of MMLV protease is known to overlap into the C-terminal Gag open reading frame (ORF) by 4 amino acid residues (T L D D) (21). Thus, the gag stop (amber) codon TAG (X in Fig. 1) is part of the protease coding sequences and can be read through by a Gln suppressor tRNA. An alignment of the protease N terminus emerged only in the way shown in Fig. 1; the L*A was provided as the r...

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Extended Analysis of the In Vitro Tropism of Porcine Endogenous Retrovirus

Cited by 197 publications

References 22 publications

Microchimerism and transmission of porcine endogenous retrovirus from a pig cell line or specific pathogen-free pig islets to mouse tissues and human cells during xenografts in nude mice

Microchimerism and transmission of porcine endogenous retrovirus from a pig cell line or specific pathogen-free pig islets to mouse tissues and human cells during xenografts in nude mice

Polymerase chain reaction in detection of porcine endogenous retrovirus (PERV) from porcine tissues

Molecular and Enzymatic Characterization of the Porcine Endogenous Retrovirus Protease

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