Transmission of Porcine Endogenous Retrovirus Produced from Different Recipient Cells In Vivo

Abstract

Humanized pigs have been developed to reduce the incidence of immune rejection in xenotransplantation, but significant concerns remain, such as transmission of viral zoonosis. Porcine endogenous retroviruses (PERV), which exist in the genome of pigs, are produced as infectious virions from all porcine cells and cause zoonosis. Here, we examined the possibility of zoonosis of hosts under conditions of immune suppression or xenotransplantation of cells producing host-adapted viruses. Upon transplantation of PERV-producing porcine cells into mice, no transmission of PERV was detected, whereas, transmission of PERV from mice transplanted with mouse-adapted PERV-producing cells was detected. In addition, the frequency of PERV transmission was increased in CsA treated mice transplanted with PERV-producing murine cells, compared with PERV-producing porcine cells. Transmission of PERV to host animals did not affect weight but immune responses, in particular, the number of T cells from PERV-transmitted mice, were notably reduced. The observed risk of PERV zoonosis highlights the requirement for thorough evaluation of viral zoonosis under particular host conditions, such as immunosuppressive treatment and transplantation with host-adapted virus-producing cells.

Fig 1. Detection of PERV genes expressed from PERV/NIH3T3 cells by PCR.

(A) PCR products of PERV pol gene by nested PCR from gDNA isolated from five cell lines (Nos. 1–5) of PERV/NIH3T3. (B) RT-PCR products of the PERV pol gene from the supernatants of five PERV/NIH3T3 cell lines. Lane Non, non-transfected NIH3T3 cells. Arrows indicate the expected size of amplified pol.

Fig 2. Percentage of PERV-positive organs from mice transplanted with PERV/NIH3T3 or PK15 in the presence or absence of CsA.

PERV-positive organs were analyzed based on genomic DNA from individual organs of mice transplanted with PERV/NIH3T3 (mPERV) and PK15 (pPERV) by PCR. *: PERV was not detected in all organs.

Fig 3. Immunohistochemical detection of PERV env protein from spleen of PERV-infected mice.

Immunohistochemistry was performed in spleens from CsA-non treated mice transplanted with NIH3T3 cells (A), non-treated mice transplanted with PERV/NIH3T3 cells (B), CsA-treated mice transplanted with PERV/NIH3T3 cells by subcutaneous injection (C), and CsA-treated mice transplanted with PERV/NIH3T3 cells by the kidney capsule method (D). Arrowheads in (C) and (D) indicate staining of PERV env protein as brown spots.

Fig 4. PCR detection of PERV pol, neomycin-resistant, and porcine mitochondrial COII genes in mice transplanted with PERV/NIH3T3 or PK15 cells.

PERV pol (200bp), neomycin-resistant (300bp), porcine mitochondrial COII (250bp), and murine GAPDH (100bp) genes were detected from gDNA extracted from PERV/NIH3T3 cells, PK15 cells, and liver tissues from Group 1 (G1), Group 4 (G4), and group 6 (G6).

Fig 5. Body weights of PERV-infected or non-infected mice after CsA treatment.

Weights of animals in each group were measured every week for 5 weeks. Each dot indicates the body weight of mice in CsA-treated (G1, G3, G5), untreated (G7, G9, G11), PERV-infected (G3, G5, G9) or uninfected groups (G1, G7, G11).

Fig 6. Effects of PERV infection on lymphocytes and CD4/8 cells in host animals.

Cells in splenocytes isolated at 7 weeks post-transplantation were stained with fluorescence-conjugated antibodies, and the number of labeled cells were measured by FACS. (A) Percentage of T and B cells in splenocytes of CsA-treated (n = 24) and untreated (n = 20) mice. (B) Percentage of CD4+ and CD8+ T cells in splenocytes of PERV-infected (n = 6) and uninfected (n = 6) mice. *: significantly different between PERV-infected and non-infected groups (Student t-test, p<0.05). The error bars indicate standard deviations.