[Highly efficient inhibition on replication of grass carp reovirus mediated by chemically synthesized small interfering RNAs].
Two short interfering RNAs (siRNA-RdRp1286, siRNA-RdRp1441) and one short interfering RNA (siRNA-OCP117), targeted to the RNA dependent RNA polymerase (RdRp) gene and outer capsid protein (OCP) gene of Grass carp reovirus (GCRV) respectively, were chemically synthesized and transfected into the CIK cells by lipofectamine 2000. 6 hours after transfection, the transfected CIK cells were challenged with GCRV. The culture media were collected at 48h post challenge and the virus was titrated in microculture system to evaluate the inhibition effect on GCRV replication mediated by siRNAs. Referring to the mRNA level of housekeeping gene beta-actin, RT-PCR was applied to detect the level of GCRV mRNA in transfected and challenged CIK cells. The results showed that the viral titer (lgTCID50/0. 1mL) in siRNA-RdRp1286, siRNA-RdRp1441 and siRNA-OCP117 transfected CIK cells were 4.41 +/- 0.16, 3.83 +/- 0.44 and 1.94 +/- 0.42 respectively, which were significantly lower than that in virus infection positive control (7.92 +/- 0.52) (P < 0.01). No significant change in viral titer was observed in the group transfected with siRNA negative control after challenged with GCRV (7.50 +/- 0.17, P > 0.05). Compared with the mRNA transcriptional level of beta-actin gene in virus infection positive control, the mRNA levels of GCRV in siRNA-RdRpl 286, siRNA-RdRp1 441 and siRNA-OCP117 transfected CIK cells were reduced significantly and the inhibition rate reached to (82.08 +/- 2.15)%, (89.19 +/- 1.14).% and (92.62 +/- 0.17)%, respectively. The mRNA level of GCRV in the siRNA negative control group had no noticeable change (P > 0 05).
Li
Bing
B
Fishery College, Huazhong Agricultural University, Wuhan 430070, China.
Fan
Yu-Ding
YD
Li
Yan-Qiu
YQ
Xu
Jin
J
Zhou
Yong
Y
Zeng
Ling-Bing
LB
D000818:Animals; D002347:Carps / Q000821:virology*; D002478:Cells, Cultured; D034741:RNA, Small Interfering / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D012087:Reoviridae / Q000187:drug effects* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014779:Virus Replication / Q000187:drug effects*
D034741:RNA, Small Interfering