Establishment and Characterization of Molecular Clones of Porcine Endogenous Retroviruses Replicating on Human Cells

Abstract

The use of pig xenografts is being considered to alleviate the shortage of allogeneic organs for transplantation. In addition to the problems overcoming immunological and physiological barriers, the existence of numerous porcine microorganisms poses the risk of initiating a xenozoonosis. Recently, different classes of type C porcine endogenous retoviruses (PERV) which are infectious for human cells in vitro have been partially described. We therefore examined whether completely intact proviruses exist that produce infectious and replication-competent virions. Several proviral PERV sequences were cloned and characterized. One molecular PERV class B clone, PERV-B(43), generated infectious particles after transfection into human 293 cells. A second clone, PERV-B(33), which was highly homologous to PERV-B(43), showed a G-to-A mutation in the first start codon (Met to Ile) of the env gene, preventing this provirus from replicating. However, a genetic recombinant, PERV-B(33)/ATG, carrying a restored env start codon, became infectious and could be serially passaged on 293 cells similar to virus clone PERV-B(43). PERV protein expression was detected 24 to 48 h posttransfection (p.t.) using cross-reacting antiserum, and reverse transcriptase activity was found at 12 to 14 days p.t. The transcriptional start and stop sites as well as the splice donor and splice acceptor sites of PERV mRNA were mapped, yielding a subgenomic env transcript of 3.1 kb. PERV-B(33) and PERV-B(43) differ in the number of copies of a 39-bp segment in the U3 region of the long terminal repeat. Strategies to identify and to specifically suppress or eliminate those proviruses from the pig genome might help in the production of PERV-free animals.

Long-term follow-up failed to detect in vitro transmission of full-length porcine endogenous retroviruses from specific pathogen-free pig islets to human cells

“…These sequences are able to produce type C particles, whose release by pig cell lines [21±23] and primary cells [17,20] has been described. Full-length endogenous retroviral genome with env gene polymorphism has shown up in several pig strains [12,14], including SPF pigs [18]. Thus, the use of SPF pigs does not eliminate the risk of transmission of PERV to xenograft recipients.…”

Evolutionary Spread and Recombination of Porcine Endogenous Retroviruses in the Suiformes

“…The first two classes (7,42) productively infect human cells in vitro, thus posing a serious risk in xenotransplantation, while the latter (1) does not replicate on human cells. There are only minor genetic differences between the classes, being most prominent in the receptor-binding domain of the Env protein.…”

Developing Xenostandards for Microbiological Safety: New Zealand Experience

“…This is because the majority of PERV sequences appear to be either defective or deleted, i.e., they contain only one or two intact retro-viral open reading frames of gag, pol and env (Bosch, Arnauld, & Jestin, 2000;Czauderna, Fischer, Boller, Kurth, & Tonjes, 2000;Herring et al, 2001;Niebert, RogelGaillard, Chardon, & Tonjes, 2002). Consequently PERV proviral copy number is another important characteristic that should be investigated when assessing a donor herd for infectious risk.…”