Study On The Inhibition Of Replication Of Grass Carp Reovirus By RNA Interference

Grass carp (Ctenopharyngodon idellus) is one of the most important species in freshwater aquaculture of China, its yield accounts for20%in total aquaculture production yield. Grass carp reovirus (GCRV) is the causative agent of the grass carp hemorrhage, the most severe viral disease of fish in China. This pathogen causes huge economic losses. GCRV has been recognized by ICTV as a species belonging to the genus Aquareovirus, AQRV-C. During this years, there are also more than20strains of aquareovirus isolated from grass carp. Based on the phylogenetic relationship of these isolates, there are three genotypes, the similarity was less than30%among the three genotypes. Currently, there is no effective treatment method against GCRV infection. Progress has been made with immune based preventive strategies, but they are far from being applicable in the practical production of grass carp. Therefore, studies on new methods for the prevention and treatment of this disease are warranted. A new strain grass carp reovirus that is the true cause of the current epidemic of Grass carp haemorrhage was isolated in this study, and single-siRNA, multiple-siRNA expression vectors targeting the GCRV were constructed to inhibit viral replication. The results were present as below:1. A new grass carp reovirus strain named GCRV-104was isolated from hemorrhagic grass carp in Hubei, China and identified as Aqureovirus by the Electron microscopy and SDS-PAGE assays. The complete nucleotide sequences of genomic segment8(S8) was1319bp in length with51%G+C content and encoding VP6protein. Analysis of S8gene and amino acid sequences and Phylogenetic analysis indicated that it belongs to the family Reoviridae, genus Aqureovirus. But it was far from the other isolates reported before. All the results indicated that GCRV-104is a new strain grass carp reovirus that was a basic materal for prevention and treatment of grass carp hemorrhage.2. Two DNA constructs targeting the grass carp reovirus (GCRV) RNA dependent RNA polymerase (RdRp) gene and outer capsid protein (OCP) gene that were each64bp in length were chemically synthesized and cloned into pSilencer2.1-U6neo plasmid, named pSi-RdRp1286and pSi-OCP117, respectively. After transfection of pSi-RdRp1286and pSi-OCP117plasmids 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-RdRp1286and pSi-OCP117reduced the viral titers by5.471gTCIDso/mL and4.371gTCIDso/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 by89%and73%, respectively. These results proved that the plasmid-transcribed shRNAs could effectively inhibit GCRV replication in CIK cells. These shRNAs provide potential tools for inhibiting GCRV infection and replication both in vitro and in vivo.3. Two multiple-siRNA expression vectors were generated, named pMultisiVP2/2and pMultisiVP6/7. One targeted the VP2gene of GCRV-JX09-01and the VP2gene of GCRV-104; another targeted the VP7gene of GCRV-09-01and the VP6gene of GCRV-104. Two multiple-siRNAs expression vectors could simultaneously inhibit the expression of GCRV-JX09-01and GCRV-104without cell toxicity. Compared with the virus positive control, the GCRV-JX09-01VP2gene expression level was10.98±2.5%, while the VP7gene expression level was19.37±3.7%; the GCRV-104VP2gene expression level was10.16±3.1%, while the VP6gene expression level was13.22±2.75%. Expression of the GCRV gene in CIK cells transfected with pSiNC were not reduced compared to the positive control. These results show that pMultishVP2/2and pMultishVP6/7inhibited both GCRV-JX09-01and GCRV-104replication by silencing the gene expression. This study is the first report to apply multiple small interferring RNA (siRNA) expression system in fish cell line. The advantages of multiple-siRNA expression vector are demonstrated using a model system simulating the emergence of mutated viruses. This approach also could provide a possible therapeutic strategy against multiple virus co-infection.