Detection And Characterization Of Porcine Endogenous Retroviruses From Chinese Miniature Pigs

Xenotransplantation is considered to be a promising approach to alleviate the shortage of human donor organs. Pigs are selected as the most suitable animals. However, concerns regarding the microbial safety of xenotransplantation were raised when PERVs were shown to infect human cells in vitro. It is feared that xenotransplantation of porcine cells/tissue/organs to immunosupressed patients might cause cross-species infections of PERV and in a worst case scenario, the pandemic in the human community. It is necessary to establish long-term appraisal for the risk posed by PERV in xenotransplatation. Studies on PERV biology and potential for pathogenicity to increase our understanding of the risk in pig to human xenotransplantation have become a new research focus.Chinese miniature pigs are the potential organ donors for pig-to-human xenotransplantation. Studies on xenotransplantation have been carried out and transgenic porcine skin for pig to human xenotransplantation is in industrialization. However, so far, in China, an adequate level of information on PERV from Chinese miniature pigs has not been available. To expore the resourses of Chinese miniature pigs and to provide safe organ donors for xenotransplantation, it's essential to do further studies on PERV on the basis of our previous studies. In this study, we intended to estimate copy numbers of PERV integrated in the host genome. This will be helpful for screening donor pigs with few copies of PERV and be beneficial for pig to human xenotransplantation. Besides, we intended to do studies on the characteristics of PERV from Chinese miniature pigs and tried to learn the molecular effect of human-tropic PERV on human cells. These studies will be helpful for revealing PERV potential pathogenesis and for the safety appraisal in xenotransplatation. The studies include the following:(1) Detection of copy numbers of porcine endogenous retrovirus from Chinese miniatureThe oligonucleotide primers used for real-time amplification of PERV were designed from the polymerase (pol) gene. Real-time PCR was performed to detect the copy numbers of PERV integrated in the host genome.The copy numbers of PERV from WZSP genomic DNA was in the range of 1.38±0.33 to 14.23±3.31, while the copy numbers of PERV from BMP genomic DNA was in the range of 2.96±0.76 to 58.46±3.50 (Table 3). The statistical analysis showed significant differences between these two breeds (P<0.01; Wilcoxon 2-Sample Test). The copy numbers of PERV from WZSP genomic DNA was less than that from BMP genomic DNA.The PERV sequence load was lower in the genome of WZSP. Thus WZSP with few copies of PERV could probably be screened by selecting breeding. Then miniature pigs without intact PERV proviruses could be screened and PERV might be knocked out. Thus the elimination of PERV could be achieved. Therefore, compared with other breeds, WZSP has the potential to be a favorable choice for xenotransplantation. In addition, compared with Southern Blot, the SYBR Green I-based real-time quantitative PCR assay described here provide a simple and rapid method for estimating copy numbers of PERV integrated in the genome and could be applied to do large-scale screening. It could be an excellent tool for screening donor pig and also be helpful for evidence of PERV transmission in clinical samples from human subjects treated with porcine xenotransplantation products.(2) Cloning and characterization of full-length proviral DNA of PERV from WZSPPERV provirus was amplified in two overlapping halves. 5'and 3'fragments were isolated, subcloned and fused to generate full-length proviral DNA using the unique restriction site within the overlap between 5'and 3'halves. Full-length nucleotide sequences of PERV-WZSP and other PERVs were aligned and phylogenetic tree was constructed from deduced amino acid sequences of env. The results indicated that the sequence of PERV-WZSP was 8,899 bp long. It had intact gag, pol, env ORFs flanking by two LTRs. Sequence alignment showed that PERV-WZSP had high similarities with PERV of other origins but differences still existed. Phylogenetic analysis indicated that PERV-WZSP belongs to the same branch with other PERV-A strains, which is consistent with the result of subtyping assay. In addition, the U3 region in 5'LTR contained three 39bp repeats which suggested that PERV of Chinese origin probably have potential capacity for increased replication in host cells. PERV-C specific sequence exited in 3'LTR of PERV-WZSP, thus suggested that partial PERV-C sequence may recombine with PERV-A sequence. Nevertheless, this implication need further study on characterization of LTR of PERV provirus from Chinese Wuzhishan miniature pigs inbred. This study is helpful for further understanding the characteristics of PERV-WZSP and also important for our further study of constructing infectious clones of PERV-WZSP.(3) Construction and identification of human cell model infected with human-tropic PERVTo study the molecular effect of human-tropic PERV on human cells and explore its potential pathogenicity, we constructed human cell model infected with human-tropic PERV and screened the cells with higher RT activity. PBMCs from WZSP and HEK293 cell line were co-cultured for 24h, then PBMCs were eliminated. There was no obvious difference in the appearance, growth characteristics and refraction between HEK293 cells post coculture and HEK293 cells as control. Several assays were done to demonstrate that HEK293 cells were infected by human-tropic PERV post coculture.PCR was applied to detect the structural genes including gag, pol and env. RT-PCR was done to detect the expression of mRNA of gag, pol and env. Western Blot was done to detect Gag protein. Then RT activity assay was done to screen the infected cells with higher RT activity. The result indicated that RT activity reached the peak around the 49th day post coculture. Thus total protein of the cells around this day was prepared for the following analysis.(4) Proteomic analysis of human cells infected with human-tropic PERV Human cells didn't show any difference in cell morphology and growth after infection by PERV. In order to reveal the molecular effect of human-tropic PERV on human cells, and to explore the potential pathogenic effects of human-tropic PERV which may be similar with other C-type retroviruses, proteomic analysis was done to study the differences of protein profile after infection of human cells by human-tropic PERV.The protein profiles of infected HEK293 cells and uninfected control were compared and analyzed by 2-DE. Ten differentially expressed proteins were identified by HDMS, including 6 upregulated proteins and 4 downregulated proteins. Real time RT-PCR and Western Blot were applied to confirm change of these proteins at the mRNA level and protein level, respectively. These differentially expressed proteins are closely related to signal transduction, cell apoptosis and protein synthesis. These identified proteins provide a number of clues and potential links to understanding the molecular effect of the infection by human-tropic PERV. This study will facilitate the appraisal of PERV in xenotransplantation.