| The use of nonhuman species as sources of organs for humantransplantation, i.e., xenotransplantation, is considered a potential solution tothe shortage of human organs and tissues for transplantation. Although tissueengineering and technology of stem cell provide a valuable soulution fororgan shortage, there are many difficultes in the application of clinicla trials.No evidence indicated the construction of a mature and funtional organ invitro by the use of tissue engineering and stem cell technology.Nonhuman primate was once considered the best source of xenograftbecause of its close relationship to human. However, nonhuman primatebelongs to distinct species and could not be largely used in clinical trials.Most importantly, the report that human immunodeficiency virus (HIV) wasorginated from a kind of primate (African green monkey) denied the use ofnonhuman primate as the source of xenograft.Pig has now been considered as the suitable source of xenotransplantation.Our results indicated that the major functions of pig liver and kidney arecompatible with that of human. There two major abstacles preventing the pigorgan to clinical trials, the first is the hyper actue rejecion, acute rejection andchronic garaft dysfunction, the transgenic (hCD55/CD59) pig would prevent the hyper acute rejection and the innovate immunosuppression could controlthe acute rejection. The second obstacle is the xenozoonsis, which is thatpathogens in the pig would transfer to human and induce infection. Thepathogens could induce the species transmission incules bacteria, fungi,parasites and virus. Pigs that are specially bred and housed can be maintedlargely free of pathogens that may prove pathogenic in human. Of greaterconcern are porcine endogenous retroviruses (PERV), which are carried inthe porcine germline and cannot be eliminated by the usual means ofpathogen-free housing. PERV, that is reside in the pig genome and showsimilarities to C-type viruses of other species were described almost 30 yearsago. A total PERV gene includes gag, pol, env and 5', 3' long terminal repeat(LTR). The products encoded by gag are core protein with many highlyconserved domains, which is called group specfic antigen. While the pol geneencodes various enzymes essentially important in virus life cycle, includethree important enzymes: protease, integratase and reverse transcriptase. Envgene encode envelope proteins determine the host tropism of the virus. LTRfunctions as the regulator of transcriptional.Recent studies have show that PERV released from porcine kidney celllines (PK15), from mitogenically activated porcine PBMC, or from porcineaortic endothelial cells are capable of infecting human cells in vitro.Approximately 50 proviral integration sites exist in the genome of differentpig breeds. There is evidence that although porcine retroviruses were unableto cause a productive infection in murine cells, they were able to rescue thereplication-defective Kirsten murine sarcoma virus from mouse, rat, andmink cell lines that were nonproducers. In theory, PERVs share the pathogenic potential of retroviruses in general, which includes insertionalmutagenesis and immunosuppression by themselves or after recombinationwith human retroviruses. Given the possibility that PERVs could betransmitted to a human xenotransplant recipient, it is further possible thatsuch an infection could be passed from a patient to close contacts and even tothe general population. Wilson et al have demonstrated that mitogenicactivation of primary PBMC from NIH pig and Yucatan pig, results in therelease of type C retrovirus that infects human cells (7). Host range analysisindicated that these viruses were distinct, as the Yucatan-derived virusinfected mink cells but the NIH miniature pig-derived virus did not. Theseexperiments suggested that PERV released from different pig species mighthave different host-cell tropism.Recently, three distinct classes of full-length PERV sequences, designatedPERV-A, -B and -C, were found in the pig genome. PERV-A, -B and -Cdisplay high sequence homology in the genes for group-specific antigens(gag) and polymerase (pol) but differ in the envelope (env) genes, whichdetermine the host range. Aside from pig cell lines, PERV-A and -B caninfect several human cell lines in vitro, but PERV-C can replicate only inporcine cells. Gentic recombination has important implications in the risk ofPERV zoonosis. A recombinant isolate, PERV-A 14/220, was originallyisolated by transmission from activated PBMC from a miniature pig tohuman embryonic kidney 293 cells. PERV-A 14/220 infects human cells witha significantly higher titer than previous PERV-A and -B isolates. Anotherstudy demonstrated that the envelope region encompassing the proline-richregion is required for binding to cells in addition to the putative variable A and B. The region in the C-terminus of the SU that alters the binding andinfectivity properties of PERV-C differs only nine residues from theanalogous region of PERV-A.Initial retrospective studies on patients who had been transplanted withporcine tissues revealed no detectable transmission of PERV. Likewise, in asurvey of 160 patients who had been treated with various living pig tissues,no conclusive evidence of pig-to-human transmission of PERV was found,although persistent microchemerism was observed in 23 patients for up to 8.5years. Tests based on DNA, RNA and protein technology to provide evidenceof pig-to-human PERV transmission have been performed on recentrecipients of living pig tissue or organs, as yet, no pig-to-human transmissionhas been observed, but concern remains. In contrast, a recent study utilizingNOD/SCID mice revealed PERV infection in several tissue compartmentsafter transplantation of pig pancreatic islets, indicating the xenozoonoticpotential of those retroviruses. Furthermore, there has been no evidence ofdisease transmission in the nonhuman primate xenotransplant studiesperformed to date.Animal model studies demonstrate that preimmunization of mice withPERV virons purified from porcine cells generates an immune response thatinduces the rejection of porcine cellular xenografts. Furthermore, porcinethyoid xenografts were more rapidly rejected after immunization with PK15cells than with pig PBL, and this did not correlate with higher class I MHClevels but with production of more PERV RNA and efficient in vitroproduction of PERV virions by the PK15 cells. Both conventional rejection innonimmunized mice and PERV virion-induced accelerated rejection were prevented after in vivo depletion of peripheral CD4 T cells, indicating thatboth types of rejection are CD4 T-cell depedent. The results showed thatPERV xenoantigens can function as important transplantation antigens andcan target cellular xenografts for accelerated rejection in preimmunized hosts.PERV-A,-B existed in all tested Chinese pigs, and no PERV-C wasdetected. BMI genome had more copies of PERV-gag, pol, envB gene thanother pigs, which indicated copy number of PERV increased duringinbreeding. The average concentration of reverse transcriptase in BMI wasfar lower than that of HIV-1 and PK15, indicating the low infectivity ofPERV. We also examined a few aspects of the biology of PERV infectedhuman cells by 6 months culture, and the results suggested PERV had noapparent effect on human cells. Human cells showed no acute changes ofgrowth after PERV infection. No significant effects were showed ontranscription of HERV-K genes after six-month infection with PERV, and themutation of LTR region after PERV infection had no apparent significance,and these results provided more safety information to xenotransplantation.Results of nude mice model indicated PERV could transmit from pig to nudemice, but no evidence of human cell infection was detected.This report describes the isolation and analysis of PERV pol, gag and envsequences from three Chinese pigs, together with the data from GenBank ofother viruses belong to mammal type C, and lent virus. Our focus here is onthe possible relationship between PERV and other pathogenic retrovirus topredict the possible pathogenesis of PERV. In this experiment, we designsmall interference RNAs from the long terminal repeat of PERV and studythe inhibition effect of these siRNAs. We use the small interference RNA designed from PERV gag and pol genes, based on the sequence specificdegradation of target mRNA, to study the inhibition effect of PERVexpression in pig kidney cell PK15. This study is focus on the PERV analysisfrom different tissues of Chinese minipig inbred. The results are acquiredfrom these studies as following:1. PERV gag, pol and env genes are integrated in all test tissues andorgans of BMI, including flesh, kidney, liver, spleen, intestine, lung, pancreas,skin, heart, thoracic aorte, abdominal aort, uriary bladder, esophagus, thyroidgland, ileum, trachea and gallbladder. The subtype of PERV in these tissues isPERV-A, -B, and no PERV-C is detetcted in all test tissues;2. The tropism differentiation of 12 BMI was analyzed by the use of cellco-culture. The PBMCs isolated from BMI, after being stimulated by PHAand IL-1, were lethally radiated and co-cultured with human kidney cell line293. The result showed that PERV from 6 BMI could successfully infected293 cells, while the other six could not;3. In bioinformatics study on the pol gene of PERV. It was found that thepol gene from three chinese pigs shared the same domain with somepathogenic type C, -D retrovirus, as well as lentivirus (HIV-1);4. In gag gene analysis, it was found the PERV-BMI belong to a novelgroup, it is related to several other type C retroviruses such as MuLV, FeLVand BaEV. There was also a highly conserved motif in all used retrovirus;5. The bioinformatics analysis on env reveled that there existed twoconserved sequences in BMI/WZSP-envA and the some type C retroviruses(SSAV, GALV, MuLV, FeLV and FcEV). Type C and D analysis indicatedthat envB shared some homology With KoEV, MuLV, MuSFV, MuEV, SSAV and PyEV), there existed two converved regions, one is nearly perfect match,the other is highly homology;6. The phylogenetic tree was constructed by the neighbour-joining method.The results showed some phylogenetic relationship between PERV and someother type C and D retroviruses. This may indicate a pool of common type Cretrovirus in verterates created by interspecies transfers during vertebrateevolution;7. The LTR sequence analysis was performed by the alignment of thePERV-LTR from three chinese pigs and other PERV reported in GenBank.The results showed that PERV-LTR from BMI and WZSP were highlyhomology to PERV-A and PERV-B. And the different of times of repeatsequence in these pigs indicated the differences in infection ability; whilePERV-NJP showed higly homology with PERV-C;8. Specific small inteference RNA (siRNA) target the PERV gag, pol andLTR were designed based on the aboved homology regions, which weretransfected by the liposome to suppress the expression of PERV in PK15 cells.The siRNA from designed from the LTR showed no inhibition effect, whilethe siRNA target the gag and pol, especially P2 and G1, could inhibit thePERV expression. And the inhibiton effect was to the most 48 hours posttransfection and was dosage dependent.In this report, we evaluated 17 tissues from Chinese BMI for the presenceof PERV. All examined tissue samples were PERV positive. PCR analysisrevealed that the envelope was class A and B. we have pursued an extendedanalysis of the host range variation of the PERV isolated from primaryPBMC of 12 BMI. We examined the cocultured 293 cells and found that six 293 groups were PERV-positive while the other six groups werePERV-negative. Our study firstly demonstrated that an endogenous retrovirusthat was directly infectious to the same human cells might have variation indifferent individuals of the same pig species. In the sequence analysis of bothgag and pol, it was revealed that PERV, either from Chinese pigs or othercountries, are closely related to other type-C and -D retrovirus and lentivirus,such as MuLV, BaEV, GALV, HERV, HIV and FeLV. This may indicate apool of common type C retrovirus in vertebrates created by interspeciestransfers during vertebrate evolution. In LTR analysis, the times of repeat boxwas different in analyzed PERV from both Chinese pigs and other PERVsfrom GenBank. It seems that the variation of repeat boxes in Chinese pigs hasan impact on the transcriptional activity of the LTR and thus on the level ofreplication capacity in these pigs. In this experiment, the shRNA designedfrom two functional genes of PERV (gag and pol) and LTR was used as a firststep in identifying a candidate inhibition sequence in in vivo research. Ourresults indicated that 21-nucleotide siRNAs promote PERV mRNAdegradation of the siRNA-induced silencing complex that mediates specificcleavage of target RNA. Our sutdies demonstrated that siRNA technologycan be used to supress PERV expression in its virla life cycle. Using syntheticshRNAs corresponding to different parts of the viral genes gag and pol,several suitable target regions for RNAi within the PERV genome wereidentified, among them the P2 sequence proved to be the most effective inreducing PERV expression and the silencing effect showed dosage dependentto some extent.The risk that any xenograft recipient will become infected with PERV is likely to be a function of several factors associated with the source animal,xenotransplant technique, and the recipient's characteristics, so defining therisk will be complex. If transmission of PERV does occur clinically, twooutcomes are possible—either the retrovirus is pathogenic but is restricted tothe recipient or the virus (pathogenic or not) could infect people notparticipating in the xenotransplantation process.The observations in our study, along with the results performed in ourgroups indicated that PERV transmission from pig organs or tissues torecipient and caused pathogenic changes in vivo seems only a remotepossibility. However, the fact that PERV showed some relationship to C-type,D-type retrovirus and lentivirus warned us that the latent pathogenicity ofPERV could be completely negalected.The successful pig-human xenotransplantation may result in long termexposure to replication competent endogenous retrovirus. Sensitive exclusionof PERV transmission in pig to human xenotransplantation is vital to care forthe safety of transplant patients and contact persons. In general, the numberof active PERV copies is probably dependent on the particular strain ofanimal. If the functional copies in the pig genome are limited, the cloning ofPERV-free pigs would become possible. We hope that the data presented herewill be valuable for the comparison of PERV distribution in different pigsbreeds in an endeavor to rule out potentially infectious proviruses.
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