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.
Virol J. 2011 Sep 7;8:428. doi: 10.1186/1743-422X-8-428.
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.
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.
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.
靶向整合素 αv 亚基的短发夹 RNA（shRNA）是口蹄疫病毒（FMDV）的受体，在病毒附着于易感细胞中发挥关键作用。我们构建了一个 RNAi 慢病毒载体 iαv pLenti6/BLOCK-iT™，该载体在 PK-15 细胞上表达靶向 FMDV 受体、猪整合素 αv 亚基的 siRNA。我们还生产了慢病毒 stock，建立了 iαv-PK-15 细胞系，使用实时 qRT-PCR 评估了 mRNA 的基因沉默效率、间接免疫荧光检测（IIF）和细胞酶联免疫吸附试验（cell ELISA）检测整合素 αv 表达，并研究了 shRNA 在体内对 PK-15 细胞中 FMDV 复制的抑制作用。
我们的结果表明成功建立了 iαv U6 RNAi 入口载体和 iαv pLenti6/BLOCK-iT 表达载体。获得的病毒的功能滴度为 1.0×10^6 TU/mL。与对照组和 mock 组相比，iαv-PK-15 组的 αv mRNA 表达率降低了 89.5%，而 IIF 和 cell ELISA 则清楚地表明实验组的抑制作用。因此，iαv-PK-15 细胞可使病毒生长减少 3 倍以上，当用 10^2 TCID50 的 FMDV 攻击细胞时，病毒滴度降低 99%以上。
iαv-PK-15 细胞被证明是一种用于抗 FMDV 效力测试的细胞模型，本研究表明 shRNA 可能是控制 FMD 感染和传播严重程度的可行治疗方法。
