Development and validation of a western immunoblot assay for detection of antibodies to porcine endogenous retrovirus

Background Reports that pig endogenous retrovirus (PERV) infects human cell s in vitro have heightened the importance of molecular and serologic monito ring of xenograft recipients for evidence of infection with PERV, We report the development and validation of a PERV-specific Western immunoblot assay for the diagnostic testing of porcine xenografts recipients. This assay is based upon the serological cross-reactivity observed between PERV variants capable of infecting human cells in vitro and other mammalian C type retro viruses, Methods and Results. Strong reactivity between PERV expressing embryonic pi g kidney PK-15 cells and antisera raised against whole virus preparations o f murine leukemia virus, gibbon ape leukemia virus (GALV), and simian sarco ma-associated virus was demonstrated by an immunofluorescence assay, sugges ting specific antigenic cross-reactivity between this group of viruses and PERV, Western immunoblot analysis demonstrated that anti-GALV antisera reac ted with three proteins in PK-15 cells having molecular masses of 30, 55, a nd 66 kDa. Antisera specific for the Gag proteins of either GALV or simian sarcoma-associated virus reacted with the 30-kDa (major) and 55-kDa (minor) proteins present in PK-15 cells and in PERV-infected 293 human kidney cell s, likely representing reactivity to the processed and precursor forms of t he PERV Gag protein, respectively. No reactivity was seen in uninfected 293 cells. Analysis of plasma samples from 200 United States blood donors and from 58 human immunodeficiency virus-1, 18 human immunodeficiency virus-a, 13 human T-cell lymphotrophic virus-I, 21 human T-cell lymphotrophic virus- II, and 15 cytomegalovirus infected controls were negative. Conclusions. As this assay is based on PERV antigen derived from infected h uman cells, it clearly has the capacity to detect a serologic response towa rds PERV variants that have zoonotic potential and will allow for the accur ate determination of PERV-specific seroreactivity in porcine xenograft reci pients.