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
Authors: Costa Michael Rodrigues , Fischer Nicole , Gulich Barbara , Belschner Hanna , Gronewold Antonia , Tönjes Ralf R. 
Published: April 2014

Porcine endogenous retroviruses (PERV) pose a xenozoonotic risk when applying pig organs, tissues and cells in clinical xenotransplantation. Three classes of replication‐competent PERV were described in host range and interference studies [1]. Polytropic PERV‐A and PERV‐B are able to infect not only human cells but also various cell lines in vitro using host membrane proteins as receptors [2]. Although cell tropism of ecotropic PERV‐C is mainly restricted to porcine cells, PERV‐C represents a considerable infectious risk. On the one hand, PERV‐C serves as template for recombination with PERV‐A resulting in highly infectious human‐tropic PERV‐A/C [3]. On the other hand, PERV‐C may evolve towards an infectious human‐tropic variant since the PERV‐C receptor‐binding domain could be bound to human cells and solely four amino acid exchanges in the surface unit of the envC gene are sufficient to permit receptor‐mediated membrane fusion and virus entry [4]. To guarantee retroviral safety in pig‐to‐human xenotransplantation generation of appropriate pigs free from replication‐competent PERV‐A and PERV‐B as well as ecotropic PERV‐C is required.

Using PCR‐based methods and directional cloning strategies, we cloned and characterized PERV‐C. Genomic DNA was prepared from peripheral blood mononuclear cells derived from three different pig subspecies which are either already used or intended for clinical xenotransplantation. One PERV‐C clone was reconstructed using genomic DNA of an Auckland Islands pig derived from a DPF herd in New Zealand. Islet cell clusters of these PERV non‐transmitters are used in clinical trials to treat diabetes type I patients. Moreover, the potential of brain cells from these animals to treat Parkinson's and Huntington's disease is currently tested clinically. Similarly, a PERV‐C clone was reconstructed from genomic DNA of Göttingen minipig which is a PERV non‐transmitter as tested in co‐cultures with susceptible human HEK 293 cells [5]. Finally, a bacteriophage lambda library was constructed from genomic DNA of a d/d haplotype miniature pig. The individuals of this pig line show lower PERV transmission levels in vitro [6]. We isolated a λ‐clone containing PERV‐C 5′‐LTR, gag, pro/pol as well as large part of the envC gene compared to replication‐competent PERV‐C(1312) [7]. The provirus is truncated due to our cloning strategy. Nonetheless, using PCR and PERV‐C specific primers the missing part of the envC gene and 3′‐LTR was amplified and ligated to the proviral fragment present in the λ‐clone. Replication‐competence of reconstructed full‐length viruses is currently tested in susceptible ST‐Iowa cells.

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