Polymerase chain reaction assays for the diagnosis of infection with the porcine endogenous retrovirus and the detection of pig cells in human and nonhuman recipients of pig xenografts.
BACKGROUND
Pigs offer an unlimited source of xenografts for humans. However, recipients of pig xenografts are inevitably exposed to the porcine endogenous retrovirus (PERV), which is carried in the pig germline. The ability of PERV to infect human cells in vitro has heightened safety concerns regarding the transmission of PERV to pig xenograft recipients.


METHODS
In response to the need to establish laboratory tests for the surveillance of PERV infection, we have developed polymerase chain reaction (PCR) assays to detect PERV pol and gag sequences by using conserved primers and probes. In addition, we have developed a PCR assay to detect pig-specific mitochondrial DNA (mtDNA) sequences as a marker of pig cells.


RESULTS
Analysis of assay sensitivities using cloned target copies in a background of human DNA demonstrated a detection threshold of 1, 5, and 1 copy for the PERV gag, pol, and pig mtDNA PCR assays, respectively. All three PCR assays gave negative results on peripheral blood lymphocyte samples from 69 humans, as well as 6 baboons and 6 macaques, demonstrating 100% specificity. The PERV and pig mtDNA assays were integrated into a simple testing algorithm that allows the differentiation between pig cell microchimerism and true xenogeneic infection. To allow for monitoring of PERV expression, a reverse transcriptase-PCR assay was also developed to detect cell-free PERV RNA.


CONCLUSIONS
The use of the diagnostic tests described here will help define the risks of PERV transmission associated with the use of pig xenografts in humans and nonhuman primates.
Switzer
W M
WM
Division of AIDS, Sexually Transmitted Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Atlanta, Georgia 30333, USA. bis3@cdc.gov
Shanmugam
V
V
Chapman
L
L
Heneine
W
W
D000818:Animals; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis; D006801:Humans; D016030:Kidney Transplantation / Q000473:pathology*; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D012190:Retroviridae / Q000235:genetics; D012192:Retroviridae Infections / Q000175:diagnosis* / Q000635:transmission; D012680:Sensitivity and Specificity; D014183:Transplantation, Heterologous / Q000473:pathology*
D004279:DNA, Viral