title: Lentviral-mediated RNAi to inhibit target gene expression of the porcine integrin αv subunit, the FMDV receptor, and against FMDV infection in PK-15 cells.
abstract: BACKGROUND
  shRNA targeting the integrin αv subunit, which is the foot-and-mouth disease virus (FMDV) receptor, plays a key role in virus attachment to susceptible cells. We constructed a RNAi lentiviral vector, iαv pLenti6/BLOCK -iT™, which expressed siRNA targeting the FMDV receptor, the porcine integrin αv subunit, on PK-15 cells. We also produced a lentiviral stock, established an iαv-PK-15 cell line, evaluated the gene silencing efficiency of mRNA using real-time qRT-PCR, integrand αv expression by indirect immunofluorescence assay (IIF) and cell enzyme linked immunosorbent assays (cell ELISA), and investigated the in vivo inhibitory effect of shRNA on FMDV replication in PK-15 cells.
  
  
  RESULTS
  Our results indicated successful establishment of the iαv U6 RNAi entry vector and the iαv pLenti6/BLOCK -iT expression vector. The functional titer of obtained virus was 1.0 × 10(6) TU/mL. To compare with the control and mock group, the iαv-PK-15 group αv mRNA expression rate in group was reduced by 89.5%, whilst IIF and cell ELISA clearly indicated suppression in the experimental group. Thus, iαv-PK-15 cells could reduce virus growth by more than three-fold and there was a > 99% reduction in virus titer when cells were challenged with 10(2) TCID(50) of FMDV.
  
  
  CONCLUSIONS
  Iαv-PK-15 cells were demonstrated as a cell model for anti-FMDV potency testing, and this study suggests that shRNA could be a viable therapeutic approach for controlling the severity of FMD infection and spread.
authors: State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China.
authors: Jihuai
authors: J
authors: Luo
authors: Junzheng
authors: J
authors: Du
authors: Shandian
authors: S
authors: Gao
authors: Guofeng
authors: G
authors: Zhang
authors: Jingjing
authors: J
authors: Sun
authors: Guozheng
authors: G
authors: Cong
authors: Junjun
authors: J
authors: Shao
authors: Tong
authors: T
authors: Lin
authors: Huiyun
authors: H
authors: Chang
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005536:Foot-and-Mouth Disease / Q000188:drug therapy / Q000821:virology; D029544:Foot-and-Mouth Disease Virus / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects*; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D039564:Integrin alphaV / Q000235:genetics / Q000378:metabolism; D016086:Lentivirus; D008969:Molecular Sequence Data; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology*; D060888:Real-Time Polymerase Chain Reaction; D011991:Receptors, Virus / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013552:Swine; D014161:Transduction, Genetic; D053585:Virus Attachment / Q000187:drug effects*; D014779:Virus Replication / Q000187:drug effects* / Q000235:genetics
chemical_list: D039564:Integrin alphaV; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011991:Receptors, Virus