Inhibition of the replication of grass carp reovirus in CIK cells with plasmid-transcribed shRNAs

Two DNA constructs targeting the grass carp reovirus (GCRV) RNA dependent RNA polymerase (RdRp) gene and outer capsid protein (OCP) gene that were each 64 bp in length were synthesized chemically and cloned into pSilencer2.1-U6 neo plasmid, named pSi-RdRp1286 and pSi-OCP117, respectively. After transfection of pSi-RdRp1286 and pSi-OCP117 plasmids into CIK cells, the inhibition of GCRV replication in the cells were detected by observing cytopathic effect (CPE), quantitating virus titers (TCID50/mL) and real-time quantitative RT-PCR analysis of viral RdRp and OCP genes. Five days after the cells were challenged with GCRV, both pSi-RdRp1286 and pSi-OCP117 reduced the viral titers by 5.47 lgTCID50/mL and 4.37 lgTCID50/mL, respectively. Compared to the positive control, CPE induced by GCRV in transfected cells was delayed and significantly less. Furthermore, the real-time quantitative RT-PCR analysis of the viral RdRp gene and OCP gene showed that the targeted gene expression were reduced by 89% and 73%, respectively. These results proved that the plasmid-transcribed shRNAs could inhibit effectively GCRV replication in CIK cells. These shRNAs provide potential tools for inhibiting GCRV infection and replication both in vitro and in vivo.

► Plasmids transcribed shRNAs targeting the RdRp gene and OCP gene of grass carp reovirus (GCRV) were initially constructed.
► Highly effective inhibition on the replication of GCRV in cells mediated by plasmid transcribed shRNAs were determined by cytopathic effect observation, viral titration and real-time PCR detection of GCRV genes.
► The technology shown in this paper may represent a potential approach to control the grass carp hemorrhage, such as the research on the generation of antiviral grass carp species mediated by RNAi technology.