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Yucatan SLA D/D haplotype miniature swine are candidates as organ donors for xenotransplantation. A full-length replication-competent PERV-C provirus was characterized. The provirus was chromosomally mapped in the pig genome. In vitro , the virus showed increased infectivity compared to other functional PERV-C isolates. Data may be used for targeted knockout to generate PERV-C free founder animals.

Body fluids and tissues in eight doxepin (Dox)-related deaths were investigated in order to prove whether the individual concentration of Dox, the concentration sum of parent drug and its active metabolite N-desmethyldoxepin (NDox) or the concentration ratio Dox/Ndox valuably contribute to making a cause of death determination. Individual case histories were shortly described. Dox and NDox concentrations were determined by LC-MS/MS. Dox concentration measured from two cases was well within a concentration range considered therapeutic, whereas subtherapeutic dosing may have occurred in another two cases. There were two cases of fatal Dox ingestion, as well as a case of high dosage and advanced putrefaction, respectively. The liver concentration sum may be more useful if a fatal ingestion cannot be clearly separated from a person's medication usage. High concentrations could be observed in lung tissue, and combined concentrations of Dox and NDox may also be helpful in making a cause of death determination. There was a trend to a higher concentration sum in the brain with increasing combined levels in blood. Overall, the sum of the absolute figures allows a more accurate interpretation in Dox-related deaths as compared to the molar concentration ratio which may be helpful in acute ingestion. Determination of the N-desmethyl metabolite along with its parent is recommended and analysis should include more than a single specimen.

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 Gottingen 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. References [1] Takeuchi Y, Patience C, Magre S et al. Host range and interference studies of three classes of pig endogenous retrovirus. J Virol. 1998; 72: 9986–9991. [2] Ericsson TA, Takeuchi Y, Templin C et al. Identification of receptors for pig endogenous retrovirus. Proc Natl Acad Sci U S A 2003; 100: 6759–6764. [3] Wilson CA, Wong S, Vanbrocklin M, Federspiel MJ. Extended analysis of the in vitro tropism of porcine endogenous retrovirus. J Virol. 2000; 74: 49–56. [4] Argaw T, Figueroa M, Salomon DA, Wilson CA. Identification of residues outside of the receptor binding domain that influence the infectivity and tropism of porcine endogenous retroviruses. J Virol. 2008; 82: 7483–7491. [5] Semaan M, Rotem A, Barkai U, Bornstein S, Denner J. Screening pigs for xenotransplantation: prevalence and expression of porcine endogenous retroviruses in Gottingen minipigs. Xenotransplantation. 2013; 20: 148–156. [6] Oldmixon BA, Wood JC, Ericsson TA et al. Porcine endogenous retrovirus transmission characteristics of an inbred herd of miniature swine. J Virol. 2002; 76: 3045–3048. [7] Preuss T, Fischer N, Boller K, Tonjes RR. Isolation and characterization of an infectious replication-competent molecular clone of ecotropic porcine endogenous retrovirus class C. J Virol. 2006; 80: 10258–10261.