Unique Small Pig Endogenous Retrovirus Host Range And Cell Tropism

Xenotransplantation is defined as the transplantation of viable cells, organs or tissues between species, which offers a great promise for overcoming shortage of human organs for allogenic transplantation. Pigs are regarded as suitable alternatives for human cells, tissues and organs sources because of organs size, matching physiology, as well as matters of ethical. However, porcine endogenous retroviruses (PERV), which are released by normal pig cells and can infect human cells in vitro and severe combined immunodeficiency (SCID) mouse in vivo, stimulated an universe discussion about the microbial safety. For the fear of transplantation of pig derived cells/ tissues/organs into a human recipient may cause it transmit into the community and then leads the universe transmission of amphixenosis and induce tumors or immunodeficiencies. In view of the previous indentified human immunodeficiency viruses (HIV) and severe acute respiratory syndrome coronavirus (SARS-CoV) both are zoonosis and had caused life-threatening diseases, the study on the infection in pig-to-human transplantation and to assess the risk of possible PERV transmission become a focus of concern in this field.Though PERV transmission could not be demonstrated in the first recipients of clinical xenotransplantation or after numerous experimental pig-to-non-human primate transplantations, as well as inoculation of immunosuppressed small animals, the risk of transmission of PERV should not be ignored, for PERV is closely related to other mammalian type-C retroviruses, suggesting the potential for PERV-mediated oncogenesis, though no evidence have been found during detection of PERV transmission. To date, several investigators have worked on the host range and cells tropism of PERV in vitro, only limited experimental data exist to identify the lines are permissive for PERV infection and replication, and on some cell line, the susceptibility to PERV has been examined with conflicting results. In the face of still uncertain risks, efforts should continue in this area. So it is important to establish a permissive cell infected modle that supports PERV replication in vitro, which is usefull for the following establishment of the PERV in vivo viral safety evaluation system for assessing PERV replication and pathogenicity, as well as finding the strategies to prevent PERV transmission or treat PERV.The PERV is difficult to isolate for the titer is too low in vitro, this challenge in the study of PERV has delayed our understanding of in vivo replication and potential for pathogenicity. The application of pseudotyed viruses provide promise for overcoming this problem. In this report, we incorporate the PERV-WZSPP Env protein into Murine Stem cell Virus (MSCV) cores to generate pseudotyped virus with the similar host range of the PERV-WZSP, and use this PERV pseudotype to infection in a broad of cell lines derived from several different small animals for initial screening for susceptibility. To date, many researchers have produced PERV pseudotypes, and applied it in the study of infection of PERV, while, in china, there is no related report. The studies include the following:At first, the PERV envelope was amplified by RT-PCR using oligonucleotide primers specific for env, which was designed with the sequence of PERV deposited in GenBank (accession no. EF133960). It contains an important Kozak sequenc in the upstream of forward primer for optimized eukaryotic translation initiation. PCR products were temporarily cloned into pGEM-T Easy Vector System by T-A cloning technique. Then excised using EcoRâ… and ligated into the similarly digested expression construct pHCMV-VSV-G from which the VSV-G coding sequence had been removed. The resulting plasmid, pHCMV-env was confirmed by enzyme digesting and sequencing analysis. Full-length amino acid sequences of Env from PERV-WZSPP and other PERV were aligned and Env phylogenetic tree was constructed. The primers used for amplifying the GFP coding sequence was designed based on the submitted nucleotide sequences of GFP genes in the GenBank database. GFP gene fragment was obtained by PCR followed by ligating into Xhoâ… - and Bglâ…¡- digested MSCVneo retrovirus expression vector, then confirmed by enzyme digesting and sequencing analysis. In this part, Sequence alignment showed that the Env amino acid of PERV-WZSPP had high homology and close relationship with the other PERV-A, but some differences still existed. The recombinant eukaryotic expression plasmid pHCMV-env and the GFP retrovirus expression vector MSCVneo-GFP were constructed and identified by enzyme digesting and sequencing analysis, which Can be used in the productionof pseudotyped virus.The pseudotype virus was generated by cotransfection of HEK293T cells with the PERV envelope wxqression vectors pHCMV-env,MSCVneo-GFP and pGag-Pol. 72 hours after infection, the Cells and supernatants were harvested 72h later and used in env expression and the viral infection assays. The supernatants were pelleted by ultracentrifugation, the HEk293T cells were infected with the concentrated dilutions of viral supernatants. DNA and total RNA from transfected cells were isolated,and then use PCR and RT-PCR to detect to integrate and expression of env gene in those cells, and the pseudotype infection was monitored inspection of expression of GFP in target cells. The result reveals that the detection of PCR, RT-PCR and inspection of expression of GFP are all positive, which demonstrates that the env gene can integrate into the HEK293T and express successfully, and the produced pseudotype virus was infectious and have efficient titer. To analyze the host range and cell tropism of PERV, pseudotype virus were produced in large scale, and then infect nine cell lines derived from different small animals relative to HEK293T positive controls. Cell culture, infect and examine protocols are similar to the above. The result indicated that PERV from WZSP could infect cell lines derived from mink and feline are likely to infection by the peseuotype virus relative to HEK293T positive controls, while cells lines derived from mouse, rat, dog, hamsters and non human primate animal African green monkey were unsusceptible to infection. The PERV pseudotype virus titer observed on Mv.1.lu and FK-81 were both lower than on HEK293T, and the Mv.1.lu exhibited a higher susceptibility to FK-81. This result suggested PERV from WZSP had limited host range compared with other PERV.At last, in order to learn more about what resulted in the variation of host range among different original PERV,we compare the Env amino sequence between PERV-WZSPP and the reported PERV-A. The result demonstrated that the Env amino acid of PERV-WZSPP which differs from the reported consensus sequence (EMBL y12238) at three amino acid ( P-to -A at position 254, E-to K at the position 266 and P-to-S at position 291), thus suggest that the difference in those three points lead the difference in host range between the two strain PERV. Nevertheless, this implication need further study on characterization of Env of PERV. This study is guidable for further understanding the molecular determinants of human cell tropism of PERV.We firstly analyze the host range and cell tropism of Chinese miniature swine via the pseudotype virus producing by bearing the PERV-WZSP Env protein onto the MSCV core, which provide clues and potential links to establish the in vivo viral safety evaluation system of PERV from Chinese miniature pigs. On the basis of difference in the human cell tropism determined molecular, critical region of tropism can be reformed through mutation, modification and mutation, which will facilitate the further study the function of those molecular. This study is helpful for the understanding of in vivo replication, potential for pathogenicity, and development of effective anti-viral or vaccines strategies to prevent transmission of PERV. Additonly, host rang and cell tropism of PERV could provide guidance in the pig to human xenotransplantation.