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Xenotransplantation of porcine organs, tissues, and cells inherits a risk for xenozoonotic infections. Viable tissues and cells intended for transplantation have to be considered as potentially contaminated non-sterile products. The demands on microbial testing, based on the regulatory requirements, are often challenging due to a restricted shelf life or the complexity of the product itself. In Europe, the regulatory framework for xenogeneic cell therapy is based on the advanced therapy medicinal products (ATMP) regulation (2007), the EMA CHMP Guideline on xenogeneic cell-based medicinal products (2009), as well as the WHO and Council of Europe recommendations. In the USA, FDA guidance for industry (2003) regulates the use of xenotransplants. To comply with the regulations, validated test methods need to be established that reveal the microbial status of a transplant within its given shelf life, complemented by strictly defined action alert limits and supported by breeding in specific pathogen-free (SPF) facilities. In this review, we focus on assays for the detection of the porcine endogenous retroviruses PERV-A/-B/-C, which exhibit highly polymorphic proviral loci in pig genomes. PERVs are transmitted vertically and cannot be completely eliminated by breeding or gene knock out technology. PERVs entail a public health concern that will persist even if no evidence of PERV infection of xenotransplant recipients in vivo has been revealed yet. Nevertheless, infectious risks must be minimized by full assessment of pigs as donors by combining different molecular screening assays for sensitive and specific detection as well as a functional analysis of the infectivity of PERV including an adequate monitoring of recipients.

Porcine cytomegalovirus (PCMV) is widely distributed in pigs and difficult to detect due to latency. PCMV infection of source pigs was associated with early graft failure after cardiac and renal xenotransplantation into nonhuman primates. Importantly, PCMV infection of the first genetically modified pig heart into a human may have contributed to the reduced survival of the patient. Sensitive and reliable assays for detection of latent PCMV infection are thus indispensable. Here, we report the development of five peptide‐induced rabbit antisera specific for PCMV glycoprotein B (gB) and their validation for detection of PCMV in infected pig fallopian tube (PFT) cells by immunofluorescence and electron microscopy (EM). The anti‐gB antibodies were also used for detection by Western blot analysis of PCMV purified from the supernatant of infected PFT cells. Sera of infected versus non‐infected pigs have been compared. In parallel, PCMV viral load in blood samples of the animals was quantified by a novel highly sensitive nested‐PCR and qPCR assay. A combination of four partly overlapping peptides from the gB C‐terminus was used to establish a diagnostic ELISA for PCMV gB specific pig antibodies which is able to differentiate infected from non‐infected animals and to quantify maternal antibodies in neonates. The combination of a highly sensitive nested PCR for direct virus detection with a sensitive peptide‐based ELISA detecting anti‐PCMV gB‐antibodies, supplemented by Western blot analysis and/or immunohistochemistry for virus detection will reliably differentiate pigs with active infection, latently infected pigs, and non‐infected pigs. It may significantly improve the virologic safety of xenotransplantation.

The infection of human transplant recipients by porcine endogenous retrovirus (PERV) is a safety issue for xenotransplantation (XTx). CRISPR/Cas9 technology has enabled the generation of pigs free of functional PERVs, and the susceptibility of these animals to reinfection by PERVs remains unclear. To assess virological safety, we characterized a cell line in which PERVs have been inactivated by CRISPR/Cas9 (PK15 clone 15) for its susceptibility to infectious PERV. First, basal expression of PERV pol, the porcine PERV-A receptor (POPAR), and reverse transcriptase (RT) activity of PERV were determined. PK15 clone 15 cells were inoculated with PERV and monitored post infection for virus expression and RT activity. Particles were visualized by electron microscopy. Our data show that PK15 clone 15 cells still produce viral proteins that assemble to produce impaired viral particles. These virions have an irregular morphology that diverges from that of mature wild type. The particles are no longer infectious when tested in a downstream infection assay using supernatants of PK15 clone 15 cells to infect susceptible swine testis-IOWA (ST-IOWA) cells. The expression of POPAR was quantified to exclude the possibility that lack of susceptibility to reinfection, for PERV-A, is caused by absence of viral host receptor(s). PK15 and PK15 clone 15 cells do, in fact, express POPAR equally. PERV RT inactivation mediated by CRISPR/Cas9 does not compromise virus assembly but affects virion structure and proviral integration. The constitutive virion production seems to maintain cellular resistance to superinfection and possibly indicates a protective side effect of this specific CRISPR/Cas9 mediated RT inactivation.

GRAB (Protein G-related alpha2M-binding protein) is a surface protein of group A streptococci and exhibits high affinity for alpha2-macroglobulin (alpha2M), a broad-range protease inhibitor. It is the sole alpha2M-binding protein of group A streptococci that has been shown to promote bacterial virulence in a mouse model of skin infection. The binding site for alpha2M was predicted to be in the N-terminal A domain of GRAB. In the present study, the alpha2M-binding domain was first narrowed down to 34 amino acids (amino acids 34-67) using variable truncated N-terminal GRAB fusion proteins. The sequence of the identified domain was used to design overlapping synthetic peptides of different sizes, which were then immobilized on a membrane and assayed for their alpha2M-binding activity. The peptide screening revealed two binding motifs of ten amino acids length, located in the DeltaA (N-terminal part of the A domain) region (amino acids 34-67) with the sequences PRIIPNGGTL (amino acids 41-50) and NAPEKLALRN (amino acids 56-65) respectively. These motifs were used for systematic mutational analysis by generating synthetic peptides containing individual amino acid substitutions at every position of the mapped binding regions. The results indicated a critical role for the arginine residue at position 42 in the first binding domain and at position 64 in the second binding region. Validation of arginine residues as the critical amino acids for alpha2M binding was achieved by site-directed mutagenesis and binding assays. Competitive inhibition assays with GRAB containing amino acid substitutions R42G (Arg42-->Gly), R64G and R42G/R64G indicated differential contribution of the arginine residues at positions 42 and 64 to alpha2M-binding activity and, thus, their involvement in GRAB-induced virulence.

Background: Decellularized human pulmonary heart valve (dhHV) scaffolds have been shown to be the gold standard especially for younger, adolescent patients.However, human heart valves are limited in availability. Xenogeneic decellularized pig heart valves (dpHV) may serve as alternative. Methods:The efficacy of DNA reduction processes upon decellularization of heart valves from German Landrace pigs was analyzed by measurements of remaining nucleic acids including proviral porcine endogenous retrovirus (PERV) sequences.Porcine pulmonary heart valves (pPHV) were decellularized by three different protocols and further treated with DNaseI or Benzonase, at varying incubation times.DNA isolated from valve associated muscle (m), valve cusp (c), and pulmonary artery (pa) was monitored by PCR and qRT-PCR using GAPDH and the PERV polymerase (pol) for read-out.Results: Decellularization of pPHV led to a significant reduction of DNA (>99%) which could be further significantly increased for (m) and (pa) by nuclease treatment, reducing proviral PERV pol from approximately 5 × 10 7 to 5 × 10 3 copies/mg in nuclease treated tissues. Conclusions:Both nucleases demonstrated comparable activities. But DNaseI revealed to be less consistent for PERV, especially at muscular tissue. Noteworthy, remaining proviral sequences are still detectable by PCR; however, due to the absence of the cellular replication machinery the production of infectious particles is not expected. Decellularization and nuclease treatment of pPHV is an efficient procedure to reduce the DNA content including PERV, thus represents a valuable option to increase virus safety independently from the source animal background. K E Y W O R D SPERV, virus safety, xenogeneic decellularized heart valve

Epigenetic reprogramming has functional impact on retrotransposons. Thus, the induction of pig-derived pluripotent cells influences their PERV expression profile. Data emphasize the necessity to focus on animals, which show non-functional endogenous viral background to ensure virological safety.