Investigation And Experimental Research Of Porcine Endogenous Retrovirus Related To Xenotransplantation

BackgroundNowadays, severe hepatitis, liver failure, and liver cancer in patients with liver disease are the leading causes of death. Liver transplantation is the most effective treatment of such diseases. But, the increasing shortage of the human organ transplantation donor has become the key factor of limiting organ transplantation, many patients lost the chances of survival with no promptly liver transplantation. At present, xenotransplantation, tissue engineering, and technology of stem cell are the main feasible ways of solveing the problem. Though tissue engineering and technology of stem cell provide a feasible solution to organ shortage, they have many difficulties in construction of the functional organs. People have made great progress in xenotransplantation by work hard for many years. Xenotransplantation, the transplantation of live animal organs, tissues or cells into recipients of another species, is a possible effective solution to alleviate the donor shortage. In view of ethical considerations, growth characteristics, organ size, and physiological characteristics, pig has been the best organ supply for researchers of xenotransplantation. Although pig-human organ transplantation has not been achieved, the pig tissue and cell as the substitute of human diseased tissue has been used gradually in clinic. But, there are two major problems:Strong immune rejection would happen in the receptors, and Viruses and bacteria can be spread between species. Knockout of pig α-1,3-galactose transferase gene have overcome immune rejection in the receptors. Pig cells, tissues, and organs all carry porcine endogenous retrovirus (PERV) and its genomic or proviral DNA, which is inherited to their offsping by Mendelian manner. Normally, with a long period of evolution for host, PERV proviral DNA in host cell mutated and thus can not synthesize protein, so the pig does not ill with PERV. However, once PERV cross the species barrier, they may proliferate and cause diseases in the host. Lieber found that mouse endogenous retroviruses, which are highly homologous with PERV, had made gibbon suffering from leukemia. PERV can not be eliminated in pigs by rising in specific pathogen-free conditions or by simple outcross breeding, so there is the potential risk of PERV dissemination between species in pig-human xenotransplantation.It has been proved that PERV is a typical mammalian C-type retrovirus with genomic structure of gag, pol, env and3’,5’ long terminal repeat (LTR). PERV is classified to two subtypes, namely, γ(1-5) and β(1-4). It has been proved that γl class possesses infection, while others remain to be proved. At present, researchers mainly study γl-PERV, which is divided into three subtypes:PERV-A, PERV-B, and PERV-C on the basis of differences in the receptor binding domain of env. PERV-A and PERV-B have been shown to infect kinds of cells such as mink, mouse and human cells in vitro, while PERV-C is restricted to two kinds of porcine cells and one kind of human cell.Since Patience for the first time reported that PERV releasing spontaneously from PK15cells could infect human cells in vitro, the potential risk of PERV transmission in the interspecies attracted widespread attention in xenotransplantation. Endothelial cells are most likely to be infected by PERV because of its direct contact with the graft in xenotransplantation. Kuddus found that PERV can infect human vascular fibroblasts and endothelial cells in vitro and that PERV was replicated in infected cells. Wison confirmed that peripheral blood mononuclear cells (PBMCs) of two kinds of pig stimulated by karyokinesis can release PERV particles, which can infect pig and human cells in vitro. Although foreign reseachers reported that PERV can infected a variety of human cell lines such as T cell lines, bone marrow cell lines, NK cell lines, kidney cell lines and so on, which are restricted to cell lines or bloods cells, there is little of research about the susceptility of solid organ cells to PERV. In the process of in vitro culture, a recombinant PERV-A/C has been demonstrated to be drived from PERV-A and PERV-C. Its infectivity for human cells is approximately500times stronger than that of PERV-A.Up to now, there is no evidence of PERV infection in human, non-human primates, and other animals receiving pig grafts. The data of PERV infection was not found in the160patients treated using porcine tissues in a retrospective review of clinical investigations. In the4.6-8years of follow-up, researchers found that21patients who had received pig pancreas transplantation were not infected by PERV. However, it should not be neglected that PERV may infect human and even induced new diseases in pig-human xenotransplantation.Based on the advantages of clear genetic background, subtle individual differences, Chinese miniature pigs are considered to be suitable transplant donors. The Chinese experimental miniature pig is one model in this regard. With the advantages of appropriate organ size, strong disease resistance, clear genetic background, stable physiological and biochemical indices, and economic affordability, the Chinese experimental miniature pig has become a promising donor source for pig-human xenotransplantation. According to a recent clinical report, PERV reverse transcriptase activity was not observed in sera from3patients treated with a porcine liver cell-based bioartificial liver from a Chinese experimental miniature pig. As we all know, the PERV copy number carried in pig breeds serves as an important reference value for potential viral infectivity and the species’ suitability as a future donor. At present, it is known that there are multiple PERV copies in most pig breeds; the copy number differs among breeds and individuals of the same breed. Due to PERV dissemination between species depending on its transcription, PERV transcription directly affects their risk of infectivity. However, data for the distribution of PERV provirus in genomic DNA and PERV expression at the mRNA level among the population of Chinese experimental miniature pig are lacking.In this study, a series of screening experiments were performed to investigate PERV epidemiology in Chinese experimental miniature pig. These data may lay the foundation for selective breeding to benefit the application of these donors to the study of xenotransplantation. Otherwise, the effects of PERV infection on human cells were detected for further understanding the susceptibility of PERV on human cells, which provides certain of biological safety evaluation for the clinical application of pig grafts.Chapter1Genetic Prevalence of Porcine Endogenous Retrovirus in Chinese Experimental Miniature PigsObjectiveA series of screening experiments were performed to investigate PERV epidemiology in Chinese experimental miniature pig, which will lay the foundation for selective breeding to benefit the application of these donors to the study of xenotransplantation.Methods We obtained ear tissues respectively from20Chinese experimental miniature pigs and then extracted its genomic DNA and mRNA.3primer pairs (env-A, env-B, and env-C) were designed and synthesized to individually detect PERV genotypes. We selected PK-15cell genomic DNA as a positive control and dd H2O as a negative control to detect PERV genotypes by using polymerase chain reaction (PCR). Meanwhile, because the pol segment is highly conserved among PERV gene sequences, it was selected as a target gene to design specific primers. The house-keeping gene porcine transferrin receptor (TFRC) gene was selected as an internal reference gene and primers were also designed to amplify its sequence. Then we detected the PERV copy number in genomic DNA by using real-time quantitative PCR and PERV expression at the mRNA level by using real-time quantitative reverse transcription PCR according to the relative quantitative standard curves for pol and TFRC based on the plasmids.Results①To detect the distribution of PERV genotypes among the genomes of Chinese experimental miniature pigs, we evaluated genomic DNA by PCR. PERV proviral DNA were found in all20samples of pig genomic DNA. PERV genotypes mostly is PERV-A/B in all tested pigs, minority of them is PERV-A/B/C.②The copy number of PERV gene was evaluated in genomic DNA by using real-time quantitative PCR. The presence of PERV proviral DNA was positively detected in all20tested samples of Chinese experimental miniature pigs. The copy number of PERV in genomic DNA ranged from3.95±0.33(copies/cell) to95.52±1.42(copies/cell).③The expression of PERV at the mRNA level, namely, the copy number of PERV cDNA, was positively detected in all20tested samples of Chinese experimental miniature pigs by using real-time quantitative reverse transcription PCR. The copy number of PERV cDNA ranged from3.66±0.33(copies/cell) to43.03±1.62 (copies/cell).ConclusionPERV proviral DNA existed commonly in all tested Chinese experimental miniature pig. PERV genotypes mainly are PERV-A/B in all tested pigs. Although both of the copy number of PERV in genomic DNA and its expression at the mRNA level are diverse among different individual of the breed, there were still some pigs with low copy numbers for PERV. Through selective breeding technology or gene knockouts, it is hopeful to obtain individuals with lower or no risk of PERV infection, which are ideally suitable for xenotransplantation.Chapter2Preliminary study of susceptibility of human cells in vitro to The Porcine Endogenous Retrovirus (PERV)ObjectiveTo research the susceptibility of four human cells in vitro to PERV.MethodsCultured human cells were infected by cell-free supernatant from PERV-produced PK15. We selected PK-15cells as a positive control and the corresponding uninfected cells as a negative control and then extracted their genomic DNA and total RNA. PCR and RT-PCR methods were performed to detect whether these target cells were infected by PERV. Meantime, P15E antigen protein was selected as a reference, then western blot analysis was used to detect whether there is the expression of PERV protein in the infected cells.ResultsHEK293cells were susceptible to PERV infection and the PERV proviral DNA of the infected HEK293cells could express at the RNA and protein level. LX-2cells, HL-7702cells, and human primary hepatocytes were resistant to PERV infection. ConclusionThe susceptibility of different organ source human cells to PERV is different. Cells of human liver were resistant to PERV infection.