Head of Section
Impact of PERV infection on the pig transcriptome
Ralf Tönjes1, Antonia Godehardt1.
1Division of Medical Biotechnology, Paul-Ehrlich-Institut, Federal Institute for Vaccines and Biomedicines, Langen, Germany
Porcine pancreatic islets and islet cells intended for non-clinical and clinical application take a central position in the field of experimental xenotransplants. The non-clinical and clinical approaches that are in place are based on micro- or macro-encapsulated islets and islet cells. For risk management, trans-species transplantation demands a sufficient microbial testing of the donor, the transplant and the recipient based on European regulatory requirements [1-2].
Porcine endogenous retroviruses (PERVs) are a focus besides the survey of common transient and resident microbial background and process derived contaminations. PERV-A and -B are present in the genome of all pigs and are able to infect human cells in vitro. PERV-C as an ecotropic virus exclusively targets pig cells and is not present in all animals. The recombinant PERV-A/C shows elevated infectious potential in vitro.
Many attempts have been undertaken to eliminate the viral background from donor pigs e.g. by selection of PERV-C free animals and the multiple knock out of PERV-A and -B via zinc finger nucleases (ZFN) that resulted in nonviable offspring. Other approaches focus on interference in order to block any transmission from donor tissue to recipient by siRNAs, immunization of the host and/or steric barriers e.g. encapsulation. Since the elimination of PERV-A and -B was unsuccessful the selection of animals free of PERV-C bearing low loads of -A and -B is a suitable choice. Nevertheless, any long term challenge of immunosuppressed patients with PERV bearing pancreatic islets and islet cells as well as the consequence of a trans-species transmission of PERV from pig to human is unknown so far. Therefore, it is essential to evaluate the potential risk and to understand how PERV expression and PERV infection affects the gene expression profile of infected cells.
Microarray platforms hereby provide sensitive and robust diagnostic tools for comprehensive analyses of gene expression besides other complementing techniques such as RNA-Seq and RT-PCR [3-6]. For this reason, a whole genome 60K Agilent array representing the known genes of the published Sus scrofa genome (NCBI Sus scrofa 10.2-assembly) was generated [7, 8]. Its functionality was repeatedly tested in a proof of concept analysis of differential gene expression of ST-IOWA cells freshly infected with molecularly cloned PERV-C (1312) versus native uninfected cells at d7, d28 and d56. Data quality was validated by RNA-Seq comparing a series of triplicates at d28 post infection. In addition, expression profiles of PERV as well as housekeeping genes were quantified by RT-PCR to merge signal intensity of arrays with the RNA copy numbers of selected genes. Retroviral PERV activity was monitored by RT-test [9, 10]. Read out and comparison of data is not fully validated yet. However, results on PERV clearly show that an infection of native PERV-C free ST-IOWA cells with PERV-C(1312) leads to productive infection. Furthermore, the expression of genomic PERV-A envelope (PERV-A env) and PERV protease/polymerase (PERV prt/pol) mRNA is upregulated significantly. Under the experimental settings, PERV-B seems to be constitutive and there are no detectable differences before and after infections. Data indicate that retroviral infection may alter the expression of resident endogenous retroviruses as shown for PERV-A env and prt/pol. As such it has to be analysed if this is a singular event or if this effect can be repeated with other PERV viruses and cell-lines.
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