| The therapeutic use of cells, tissues, and organs derived from animal as donors for xenotransplants might help to overcome the growing shortage of human allotransplants. Pigs are among the most likely source species for xenografts for clinical use because of ethical considerations, breeding characteristics, compatible organ sizes, and physiology. However, major concerns regarding infectious risk posed by the possibility of introducing new agents from the pigs into the recipient, leading to xenozoonosis, have been raised. Among many porcine pathogens that can infect humans, porcine endogenous retrovirus (PERV) has drawn considerable attention because all pig strains carry this virus. Moreover, reports in this decade indicated that PERV released from different porcine cells could infect human cells in vitro. Thus, PERV poses potential risk in the course of pig-to-human transplantation.Chinese miniature pigs are mainly for experimental use and are considered to be appropriate organ donors in xenotransplantation in virtue of their clear genetic background and analogous organ size. We performed large-scale survey of PERV in Chinese miniature pigs and found PERV existed in all the examined genomic DNA samples. Our study showed the copy numbers in genomic DNA varied in different germline but were lower in Wuzhishan pigs (WZSP), so this breed of miniature pigs is probable to be a better choice in xenotransplantation compared with other breeds.Breeding and keeping pigs under specific-pathogen-free or qualified-pathogen-free conditions is generally assumed to reduce the potential risk of transmitting exogenous viral, bacterial, fungal, and parasitic agents by xenotransplantation. However, these methods are not appropriate for avoiding the presence of endogenous retroviruses which are transmitted in the germ line.In recent retrospective analyses of xenotransplanted human patients, no evidence was found for expression, infection, and replication of PERV in the human host. Even though these reports are somewhat reassuring with regard to safety, particularly as PERV replicates even in permissive tissue culture cells to relatively low titers, it has to be stated that no long-term xenotransplantation of physiologically functional pig tissues has been achieved so far and no vascularized pig organs have been successfully used in human patients. In contrast, a recent study utilizing NOD/SCID mice revealed PERV infection in several tissue compartments after transplantation of pig pancreatic islets, indicating the xenozoonotic potential of those retroviruses. Therefore, a better researching of cellular and molecular characteristic basis on PERV genes will be helpful for understanding the virus life circle, infection mechanism and stimulating approaches to use xenotransplantation. In other countries, several infectious clones of PERV-A, PERV-B has been successfully constructed. The replication mechanism and molecular biologic characteristics have been studied. However, so far, in China construction of PERV infectious clone has yet never been reported.In this report, we constructed the infectious clone of PERV from Wuzhishan pig. This study will be beneficial for the further study of PERV. In this study, proviral DNA of WZSP-PERV was amplified separately in two overlapping halves. Based on the sequences of PERV deposited in GenBank (accession no. EF133960), PCR primers were designed to amplify the two PCR fragments (5'half and 3'half). Sal I and Xba I site was included in the primer 5'half forward and primer 3'half reverse, respectively. Nhe I site existed in the overlapped domain. Based on PCR and molecular cloning, pBluescriptâ…¡SK+-WZS-PERV was obtained and identified with PCR and restriction enzyme digestion. All together 9 clones of pBluescriptâ…¡SK+-WZS-PERV were constructed and used for screening the infectious clone. These clones were transfected into HEK293 cells. Results of PCR and RT-PCR indicated that only one of them could lead to the integration and expression of PERV gag, pol, env genes in the transfected cells, then this clone was designated WZS-PERV(2). Cell-free supernatant of this culture was harvested and used to challenge naive HEK293 cells. Identification of PCR indicated that PERV integrated into the genome of HEK293 cells. After 6 serial passaging of the virus, SYBR Green I-based real-time quantitative PCR assay demonstrated the expression of PERV mRNA in the infected cells. Relative quantitative analysis showed the differential expression of PERV mRNA in these 6 passages. SDS-PAGE and Western Blot analysis demonstrated the expression of Gag protein both in transfected and infected cells as the expected bands indicating the molecular mass of 60kDa, 40kDa, 27kDa have been observed. Moreover, cells were tested for expression of Gag protein by an indirect immunofluorescence assay using PERV-Gag monoclonal antibody. With confocal microscope, green fluorescence was observed in the cytoplasm which confirmed the expression of Gag protein. Ultrastructural analysis with transmission electron microscope showed typical viral particles were produced in the infected cells. Sequence analysis showed that the sequence of WZS-PERV(2) was 8,939 bp long. The alignment of sequences retrieved from GenBank indicated that the sequence comprised a typical 21bp (417bp~437bp)-24bp (438bp~461bp)-37bp (464bp~500bp) domain which is similar to that in the LTR of PERV A/C recombinant stain. This result indicated that PERV infectious clone constructed in this study consisted of the PERV-C LTR and PERV-A RBD, thus suggested that WZS-PERV(2) is a PERV A/C recombinant stain. This clone could infect human cells in vitro, so it's human tropic. The 21bp-24bp-37bp domain in the LTR comprised many transcription factor combining site including NF-Y, so this domain may determine the infectivity of PERV.Many Gâ†'A mutations in the sequence of PERV took place with the passaging of Wuzhishan pigs inbred. Considering APOBEC3F (pA3F) in the innate immunity, we presumed that pA3F might contribute to these mutations. However, the exact mechanism and reasons were further required.In this study, we constructed the WZS-PERV provirus gene clone successfully and firstly acquired its infectious molecular clone. The study will be helpful to screening donor pigs for xenotransplantation and also be beneficial for monitoring the transmission of PERV after pig to human xenotransplantation and would have an impact on possibility of cloning PERV-free pigs.On the basis of constructing full length gene clone of PERV provirus from Wuzhishan pigs, infectious molecular clone of PERV from Wuzhishan pigs was firstly constructed and screened. This is important for clarifying the biological characteristics of human tropic PERV, for exploring physiological and pathological effects of PERV infecting human cells, and for analyzing and evaluating its pathogen safety in pig to human xenotransplantation. With this infectious clone, PERV coding genes could be reformed through deletion, mutation, modification, thus benefitial for functional analysis of the crucial protein region and for revealing the regulatory process of PERV and its potential pathogenic mechanism. In addition, PERV infectious clone could be applied for the studies on retroviral vectors and new DNA vaccines. It's significant for the realization of pig to human xenotransplantation. |
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