Abstract
BackgroundshRNA 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.ResultsOur 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 × 106 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 102 TCID50 of FMDV.ConclusionsIαv-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.
Highlights
ShRNA targeting the integrin av subunit, which is the foot-and-mouth disease virus (FMDV) receptor, plays a key role in virus attachment to susceptible cells
RNAi is a process of sequence specific, posttranscriptional gene silencing (PTGS) in animals and plants, which is conducted using 21 to 33 nucleotides of small interfering RNA, which are homologous in sequence to the silenced gene [2]
Target gene expression of the porcine integrin av subunit constructed with an RNAi lentivirus vector and established as an iav-PK-15 cell line We constructed the iav-pENTRTM/U6 entry clone and iav-pLenti6/BLOCK-iTTM expression clone by producing a lentiviral stock and co-transfecting the optimized ViraPowerTM Packaging Mix (3.0 μg/μl) and iav-pLenti6/ BLOCK-iTTM-DEST expression construct or pLenti6/ BLOCK-iTTM-DEST expression construct (3 μg) into the 293FT producer cell line, harvested the viral supernatant, and determined the lentiviral stock titer with blasticidin
Summary
ShRNA targeting the integrin av 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, iav pLenti6/BLOCK -iTTM, which expressed siRNA targeting the FMDV receptor, the porcine integrin av subunit, on PK-15 cells. RNAi is a process of sequence specific, posttranscriptional gene silencing (PTGS) in animals and plants, which is conducted using 21 to 33 nucleotides (nt) of small interfering RNA (siRNA), which are homologous in sequence to the silenced gene [2]. DsRNA is produced in vivo or introduced by pathogens and processed into 21-23 nucleotide doublestranded short interfering RNA duplexes (siRNA) using an enzyme called Dicer, which is a member of the RNase III family of double-stranded RNA-specific endonucleases [3,4]. In addition to dsRNA, other endogenous RNA molecules have been shown to be capable of triggering gene silencing, including, short temporal RNA (stRNA) and microRNA (miRNA) [7]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
