The study protocol and standard operating procedures were reviewed and approved by the Orient Genia Institutional Animal Care and Use Committee (IACUC No. ORIENT-IACUC-11104) for monkeys, and by the National Institute of Animal Science's Institutional Animal Care and Use Committee (No. 2012-D-008) for GT-KO pigs. Cynomolgus monkeys (Macaca fascicularis; China, 4 to 5 kg; blood type A; n=2; Orient Genia, Inc., Seongnam, Republic of Korea) were used as organ recipients. Homozygous GT-KO pigs (n=2; blood type A; 5 to 7 kg; National Institute of Animal Science, RDA, Suwon, Republic of Korea) were housed in a specific pathogen-free facility to be used as organ donors [3]. Surgical procedures and immunization protocols were performed as previously described [6]. Briefly, the donor heart and kidney were transplanted into the abdomen of the recipients by anastomosis of the donor aorta to the recipient aorta and by the donor pulmonary artery to the recipient inferior vena cava (IVC) in heart transplantation and donor renal artery to recipient aorta, and donor renal vein to recipient IVC, respectively. Recipients received induction therapy with rabbit anti-thymocyte globulin 5 mg/kg/day (Genzyme, Cambridge, USA) for 4 times, rituximab 10 mg/kg/day (Roche, Basel, Switzerland) for 2 times, and Cobra venom factor 0.05mg/kg/day (Quidel, San Diego, USA)for5 times. Whole blood from the recipient monkeys was collected in K₂ EDTA tube (BD Vacutainer, Franklin Lakes, USA). Total RNA from the blood cells was extracted using Trizol reagent (Invitrogen, Carlsbad, USA). Primer sets were designed to amplify GAPDH (For: 5'-ggaatcccatcaccatcttccagg-3'; Rev: 5'-gagccccagccttctccatg-3') (NM_002046), PERV gag (For: 5'-tcaggcggtacaccccttt-3'; Rev: 5'-gatcacgtaactcagc ctcctgtaa-3') [2], and full-length conserved PERV (For: 5'-tccagggctcataatttgtc-3'; Rev: 5'-tgatggccatccaacatcga-3') [7], respectively.

The PERV gag element was not detected during the survival periods in the blood cells of recipient cynomolgus monkeys transplanted with GT-KO pig heart and kidney (Figure 1A and 1B). In contrast, it was strongly expressed in biopsied and autopsied heart and kidney xenografts from GT-KO pigs (Figure 1C). The full-length conserved PERV was also not found during the survival periods in the blood cells of the recipient monkeys (Figure 2A and 2B), although it was expressed in the xenografted GT-KO pig organs (Figure 2C).

This is the first report of a PERV transmission after pig to monkey xenotransplantation in Korea. Based on the xenotransplantation experiments, we obtained several hematological results: 1) there was no evidence of hyperacute immune rejection during the survival periods; 2) no characteristic disseminated intravascular coagulation and thrombocytopenia, and 3) stable levels of troponin I, reflecting that the function of the grafts was normal after transplantation. And both the PERV gag and full-length conserved PERV elements were not detected in the blood cells of the recipient monkeys after heart and kidney xenotransplantation, respectively. PERVs have been shown to infect human cells in vitro, and have been found to be actively transcribed, translated, and infectious in other species in vivo after the transplantation of pig tissues [5,12]. However, several researchers reported that the patients, who were screened after receiving porcine tissue, were found to be negative for PERV transmission [4,8].Thus far, there are some controversies on the risk of PERV transmission or infection following xenotransplantation. However, ongoing research on several subtypes and their characteristics is of prime importance to rule out any associated risks caused by PERVs. Although we could not find any clues on the transmission of PERVs in blood cells of recipient monkeys, it is difficult to conclude that no PERV transmission occurs during pig to monkey xenotransplantation because of short-term adaptation. Therefore, further studies are required for long term monitoring of PERV expression in recipients.