Polymerase chain reaction in detection of porcine endogenous retrovirus (PERV) from porcine tissues

Abstract

Pigs offer an unlimited source of xenografts for humans. The use of transplants from animal origin offers a potential solution to the limited supply of human organs and tissues. However, like many other mammalian species, pigs harbor porcine endogenous retrovirus (PERV), which are encoded in their genomic DNA and are assumed to have been integrated into the porcine germline. The ability of PERV to infect human cells in vitro has heightened safety concerns regarding the transmission of PERV to pig xenograft recipients. Porcine tissues were analyzed using validated assays specifi c for PERV: polymerase chain reaction (PCR) (for PERV DNA) and reverse transcriptase (RT)-PCR (for PERV RNA). PERV-specifi c gag sequences were found in the porcine heart tissue samples using DNA-PCR and RT-PCR. PCR is a rapid and specifi c test for the detection of PERV from xenografts. These fi ndings have demonstrated that the presence of both DNA and RNA forms of PERV in porcine tissues needs to be carefully considered when the infectious disease potential of xenotransplantation is being assessed.

Quantitative Estimation of Porcine Endogenous Retrovirus Release from PK15 Cells

“…Sypniewski et al (2005) and Tacke et al (2003) showed the presence of PERVs in porcine peripheral blood mononuclear cells (PBMCs) and fragments of the liver, kidney, and heart. A similar study was conducted by Prabha and Verghese (2009), authors demonstrated that PCR is a rapid and specific test for confirmation of the presence of PERVs. However, the most of these authors demonstrated only the presence of porcine endogenous retroviruses in Our results revealed that the copy number of PERV A DNA and PERV A RNA was significantly higher than that of PERV B DNA and PERV B RNA in PK15 cells.…”

Porcine Endogenous Retroviruses in Xenotransplantation—Molecular Aspects

“…Paradis et al [ 17 ] and Sypniewski et al [ 47 ] used PCR primers situated in a highly conserved region of the PERV genome, the gag sequence, to detect all PERV types in both human and porcine samples, respectively. In addition, Prabha and Verghese [ 49 , 84 ], with the use of PCR for PERV DNA and reverse transcriptase RT-PCR for PERV RNA analyses, found PERV-specific gag sequences in aortic valve, pulmonary valve, and heart muscle samples obtained from fresh porcine xenograft tissue. Kim et al [ 85 ] detected PERV gag sequences in porcine cells by PCR to assess the usefulness of the one-step extraction method compared to the phenol extraction method, whereas Wang et al [ 86 ] assessed the safety of a bioartificial liver support system.…”

“…Detection of these genes in the test material indicates the presence of PERVs, but it does not indicate which subtype of the virus is present. Generally, it answers the question as to whether the material being tested contains genetic material of PERV origin [ 23 , 47 , 49 , 84 , 85 , 86 , 87 , 88 ]. The second group consists of primers complementary to env , a gene that is characterized by a large degree of variability.…”

“…The entire RT-PCR reaction can be carried out in a single vessel. Primers that are specific for specific PERV sequences are used for this purpose [ 49 , 84 , 88 ]. The RT-PCR reaction can also be conducted in two stages.…”

“…PERVs are integrated into the porcine genome and inherited as Mendelian traits. The expression of PERVs may differ, depending on the breed of pig and the tissue [ 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 ], but the PERV DNA copy number in the whole organism is about 50 copies per haploid genome [ 54 , 55 ]. Moreover, there are variations in PERV integration sites among breeds [ 56 , 57 , 58 , 59 ].…”