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 superna