title: Construction and characterization of an infectious replication competent clone of porcine endogenous retrovirus from Chinese miniature pigs.
abstract: BACKGROUND
  Xenotransplantation from animals has been considered to be a preferable approach to alleviate the shortage of human allografts. Pigs are the most suitable candidate because of the anatomical and physiological similarities shared with humans as well as ethical concerns. However, it may be associated with the risk of transmission of infectious porcine pathogens. Porcine endogenous retroviruses (PERVs) are of particular concern because they have been shown to infect human cells in vitro. To date, researches on the molecular characteristics and potential pathogenicity of PERV are still tenuous. In this report, an infectious replication competent clone of PERV from Wuzhishan pigs (WZSPs) in China was generated and characterized. This infectious clone will contribute to studies on PERV virology and control of PERV in xenotransplantation using Chinese miniature pigs.
  
  
  METHODS
  The proviral DNA of PERV from WZSPs was amplified in two overlapping halves. Then the two fragments were isolated, subcloned and fused to generate pBluescriptαSK+-WZS-PERV recombinant clones. Screened with RT-PCR, a molecular clone of PERV designated as WZS-PERV(2) was selected. Its infectivity and replication competency were characterized in HEK293 cells by PCR, real-time fluorescent quantitative RT-PCR, western blot, indirect immunofluorescence assay as well as sequence analysis.
  
  
  RESULTS
  The ability of WZS-PERV(2) to infect human cells and produce infectious virions were shown after transfection of the clone into HEK293 cells and infection of PERV derived from this recombinant clone. The expression of Gag proteins were detected in HEK293 cells infected with the virus derived from the clone by the indirect immunofluorescence assay and western blot. The results of sequences analysis and comparison combined with the PCR based genotyping result demonstrated that the WZS-PERV(2) belonged to PERV-A subgroup. Compared with a previous proviral DNA clone of PERV (PERV-WZSP), G to A hypermutation occurred in the env gene of WZS-PERV(2) was found, whereas APOBEC proteins have the potential to inhibit the replication of a variety of retroviruses through a cDNA cytosine deamination mechanism, so we presumed these G to A hypermutation might be the contribution of porcine APOBEC3F.
  
  
  CONCLUSIONS
  Altogether, an infectious replication competent clone of PERV from Chinese miniature pigs (WZSPs) termed WZS-PERV(2) was generated, characterized and sequenced.
authors.affiliation: Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu 610041, China.
authors.forename: Silong
authors.initials: S
authors.lastname: Xiang
authors.forename: Yuyuan
authors.initials: Y
authors.lastname: Ma
authors.forename: Qipo
authors.initials: Q
authors.lastname: Yan
authors.forename: Maomin
authors.initials: M
authors.lastname: Lv
authors.forename: Xiong
authors.initials: X
authors.lastname: Zhao
authors.forename: Huiqiong
authors.initials: H
authors.lastname: Yin
authors.forename: Nian
authors.initials: N
authors.lastname: Zhang
authors.forename: Junting
authors.initials: J
authors.lastname: Jia
authors.forename: Rong
authors.initials: R
authors.lastname: Yu
authors.forename: Jingang
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D020077:Endogenous Retroviruses / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D013552:Swine; D013556:Swine, Miniature / Q000821:virology*; D014779:Virus Replication
chemical_list: D004279:DNA, Viral