Comparison of porcine endogenous retroviruses infectious potential in supernatants of producer cells and in cocultures.

BACKGROUND: Porcine endogenous retroviruses (PERV) pose a zoonotic risk potential in pig-to-human xenotransplantation given that PERV capacity to infect different human cell lines in vitro has been clearly shown in the past. However, PERV infectious potential for human peripheral blood mononuclear cells (huPBMC) has been also demonstrated, albeit with controversial results. As productive PERV infection of huPBMC involves immune suppression that may attract opportunistic pathogens as shown for other retroviruses, it is crucial to ascertain unequivocally huPBMC susceptibility for PERV. To address this question, we first investigated in vitro infectivity of PERV for huPBMC using supernatants containing highly infectious PERV-A/C. Second, huPBMC were cocultivated with PERV-A/C producer cells to come a step closer to the in vivo situation of xenotransplantation. In addition, cocultivation of huPBMC with porcine PBMC (poPBMC) isolated from German landrace pigs was performed to distinguish PERV replication competence when they were constitutively produced by immortalized cells or by primary poPBMC. METHODS: Supernatants containing recombinant highly infectious PERV-A/C were used to infect PHA-activated huPBMC in the presence or absence of polybrene. Next, PERV-producing cell lines such as human 293/5° and primary mitogenically activated poPBMC of three German landrace pigs were cocultivated with huPBMC as well as with susceptible human and porcine cell lines as controls. PERV infection was monitored by using three test approaches. The presence of provirus DNA in putatively infected cells was detected via sensitive nested PCR. Viral expression was determined by screening for the activity of gammaretroviral reverse transcriptase (RT) in cell-free supernatants of infected cells. Virus release was monitored by counting the number of packaged RNA particles in supernatants via PERV-specific quantitative one-step real-time reverse transcriptase PCR. RESULTS: Porcine endogenous retroviruses-A/C in supernatants of human producer 293/5° cells was not able to infect huPBMC. Neither RT activity nor PERV copies were detected. Even provirus could not be detected displaying the inability of PERV-A/C to induce a productive infection in huPBMC. In cocultivation experiments only non-productive infection of huPBMC with PERV derived from 293/5° cell line and from PHA-activated poPBMC was observed by detection of provirus DNA in infected cells. CONCLUSION: Recombinant PERV-A/C in supernatants of producer cells failed to infect huPBMC, whereas coculture experiments with producer cell lines lead to non-productive infection of huPBMC. PERV in supernatants seem to have not sufficient infectious potential for huPBMC. However, extensive PERV exposure to huPBMC via cocultivation enabled at least virus cell entry as provirus was detected by nested PCR. Furthermore, results presented support previous data showing German landrace pigs as low producers with negligible infectious potential due to the absence of replication-competent PERV in the genome. The low PERV expression profile and the lack of significant replication competence of German landrace pigs raise hope for considering these animals as putative donor animals in future pig-to-human xenotransplantation. Nonetheless, data imply that PERV still represent a virological risk in the course of xenotransplantation, as the presence of PERV provirus in host cells may lead to a provirus integration resulting in insertional mutagenesis and chromosomal rearrangements.

Development of sensitive methods for detection of porcine endogenous retrovirus-C (PERV-C) in the genome of pigs.

Porcine endogenous retroviruses (PERV) represent a risk for xenotransplantation using pig cells, tissues or organs. PERV-A and PERV-B are present in the genome of all pigs and both infect human cells in vitro. PERV-C infects only pig cells and it is integrated in the genome of most, but not all pigs. Recombinants between PERV-A and PERV-C were described that infect human cells and replicate at high titres. To avoid such recombinations, PERV-C positive animals should not be used for breeding animals suited for xenotransplantation. In order to detect PERV-C positive pigs, different methods were developed such as specific PCRs using different primers, a highly sensitive nested PCR and a real-time PCR allowing measurement of proviral copy numbers. The real-time PCR was found to be useful to discriminate between contamination and actual provirus copies. The PCRs were optimized and their sensitivity was determined. Screening can be started with PCR1, if the result is negative, PCR2 to PCR5 or the nested PCR should be used, if the result is positive, the real-time PCR should be used to exclude contaminations. All methods were used to evaluate the prevalence of PERV-C and to identify PERV-C free animals. Due to the risk of contamination with cells from other animals testing should be performed with blood cells, not with ear biopsies.