1. 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.
- Author
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Luo J, Du J, Gao S, Zhang G, Sun J, Cong G, Shao J, Lin T, and Chang H
- Subjects
- Animals, Base Sequence, Cell Line, Enzyme-Linked Immunosorbent Assay, Foot-and-Mouth Disease drug therapy, Foot-and-Mouth Disease virology, Foot-and-Mouth Disease Virus genetics, Foot-and-Mouth Disease Virus metabolism, Gene Silencing drug effects, Humans, Lentivirus, Molecular Sequence Data, Plasmids, RNA, Messenger genetics, RNA, Messenger metabolism, RNA, Small Interfering genetics, Real-Time Polymerase Chain Reaction, Swine, Transduction, Genetic, Virus Replication genetics, Foot-and-Mouth Disease Virus drug effects, Gene Expression drug effects, Integrin alphaV genetics, Integrin alphaV metabolism, RNA, Small Interfering pharmacology, Receptors, Virus antagonists & inhibitors, Receptors, Virus genetics, Receptors, Virus metabolism, Virus Attachment drug effects, Virus Replication drug effects
- 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.
- Published
- 2011
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