Elsevier

The Lancet

Volume 352, Issue 9129, 29 August 1998, Pages 692-694
The Lancet

Early Report
Expression of pig endogenous retrovirus by primary porcine endothelial cells and infection of human cells

https://doi.org/10.1016/S0140-6736(98)07144-XGet rights and content

Summary

Background

The risk of interspecies transmission of retroviruses during xenotransplantation is suggested by reports of pig endogenous retrovirus (PERV) released from porcine cell lines productively infecting human cell lines in vitro and of infectious PERV being released from pig peripheral blood mononuclear cells after mitogenic stimulation. Endothelial cells are the main interface between a xenograft and the recipient's leucocytes and tissues.

Methods

We have analysed pig primary aortic endothelial cells (PAEC) together with other transplantation-relevant porcine cells and tissues for expression of PERV mRNA. Release of virus particles by PAEC was monitored by reverse transcriptase (RT) activity in the medium of cultured PAEC. Infectivity for human cells was tested by co-cultivation of irradiated PAEC with the human embryonal kidney cell line HEK293 and looking for virus release from the human cells.

Findings

PAECs, hepatocytes, lung, and skin from a variety of pig strains and breeds expressed PERV mRNA. PAEC released infectious particles. Co-cultivation of PAEC and HEK293 led to productive infection of the human cells and expression of PERV types A and B.

Interpretation

Release of infectious virus from PAEC occurred without mitogenic stimulation, suggesting a serious risk of retrovirus transfer after xeno-transplantation.

Introduction

A shortage of human organs for transplantation has led to attempts at xenotransplantation. Primate donors carry the risk of transmission of potentially pathogenic retroviruses. Pigs have been considered suitable and until recently were thought to pose a low risk of transmission of retroviruses. However, Patience et al1 demonstrated the release of pig endogenous retrovirus (PERV) particles from porcine kidney-cell lines and their infectivity for human cell lines. At least two infectious variants (PERV A and B), differing in their envelope proteins, have been detected in pig heart, spleen, and kidney.2 Wilson et al extended this finding to pig peripheral blood monocytes (PBMC), which, after mitogenic stimulation, produced virus infectious for human embryonal kidney (HEK) 293 and hela cell lines.3 These findings have provoked serious debate about the safety of xenografts.4, 5, 6, 7, 8 Since porcine aortic endothelial cells (PAEC) will be the main interface between the xenograft and the recipient's leucocytes and tissues after transplantation, we decided to find out whether these cells can release PERV particles and whether pigs of different breeds or origin express endogenous retroviruses to a similar extent.

Section snippets

Culture of endothelial cells

Endothelial cells were isolated by incubating the inner layer of pig aortas or human veins with 0·2% collagenase type A for 20 min at 37°C. Detached cells were washed twice in phosphate-buffered saline and cultured in either M199 (Biochrom, Berlin) plus 20% fetal calf serum (FCS) (Life Technologies, Eggenstein) and EC growth factor (Boehringer Mannheim) or EGM 2 (Clonetics) plus 15% FCS. Cells were harvested by incubation with 0·05 mmol/L trypsin/0·02 mol/L EDTA. HEK293 cells and the

Results

To test a representative variety of transplantation-relevant porcine cells and tissues for PERV RNA expression, total RNA was prepared from primary endothelial cells, hepatocytes, lung, and skin from minipigs, Yucatan micropigs, and German landbreed pigs obtained from 12 sites in Germany, Denmark, Russia, and France. PERV pol RNA was detected in all samples (figure 1, upper line). Porcine PK15 and human endothelial cells served as positive and negative controls. To exclude contamination of

Discussion

The promise of pig xenografts suffered a disappointment when PERV were found to infect human cell lines. Transfer of infectious endogenous virus from porcine to human cell lines in vitro does not, however, closely mirror the situation in vivo. A more recent publication on primary pig PBMCs (inevitably present in xenografts) revealed the release of PERV, after mitogenic stimulation, that were infectious for human cell lines. A closer simulation of xenotransplantation is to monitor vascular

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