Cellular Model For Inhibition Of Grass Carp Reovirus Replication Mediated By Rna Interference

Grass carp (Ctenopharyngodon idellus), belonging to cyprinid family, is a most popular commercial fish species in China. Grass Carp Hemorrhage is one of the most serious diseases of grass carp, causing more than 90% mortality and huge economic loss to the aquaculture industry. At present, effective ways for prevention and treatment of the disease is unavailable.RNA interference is a mechanism of homologous genes expression silencing induced by small interfering RNAs. The phenomenon of gene silencing has widely existed in many organisms. Because of its highly specificity and efficiency, RNAi might be a new tool for sequence-specific gene expression silencing.In this study, cellular model for inhibition of Grass carp reovirus replication mediated by RNA interference was established, and a solid basis has been set up for site-specific anti-viral molecular breeding technology in fishes. The study was carried out from the following aspects.1. GCRV were propagated at large-scale in Ctenopharyngodon idellus Kidney (CIK) cells, the genomic nucleic acid of GCRV was extracted. The cDNA was obtained through reverse transcription and the targeted segment was amplified by PCR. The PCR products separated in an agarose gel were purified and ligated into a pGEM-T vector. The product of ligation reaction was transformed into E. coli competent cells. The plasmid was extracted and identified by PCR. The positive clone was sequenced and partial sequence of RNA segment in GCRV genome was obtained.2. Two short interfering RNAs (siRNA-RdRp1286, siRNA-RdRp1441) and one short interfering RNA (siRNA-OCP117) targeted to RdRp and OCP gene of GCRV respectively were chemically synthesized and transfected into CIK cells by lipofectamin 2000.6 hours later, the transfected CIK cells were challenged with GCRV.48 hours post challenge, the culture medium was titrated in microculture system to evaluate the inhibition effect on GCRV replication. Compared with the mRNA level ofβ-actin housekeeping gene, the challenged CIK cells were collected and analyzed by RT-PCR to determine the transcriptional level of GCRV mRNA. The results showed that the titer (TCID50/0.1mL) of GCRV in culture medium of CIK cells transfected with siRNA-RdRp1286, siRNA-RdRp1441 and siRNA-OCP117 were 104.41±016, 103.83±0.44 and 101.94±0.42, which were significantly lower than that in virus infection positive control group(107.92±0.52)(P<0.01). Compared with virus infection positive control, the mRNA transcriptional levels of GCRV in CIK cells transfected siRNA-RdRp1286, siRNA-RdRp1441 and siRNA-OCP117 were reduced significantly and the inhibition rate reached to 82.08±2.15%,89.19±1.14% and 92.96±0.17%, respectively. The mRNA transcriptional levels of GCRV in negative control group had no noticeable change (P>0.05). These results show that RdRp-targeted and OCP-targeted siRNAs can effectively and specifically inhibit GCRV replication in vitro.3. Three plasmid expressed siRNAs targeted RdRp and OCP genes of GCRV were designed.3 plus-strains and 3 minus-strains of oligo-deoxynucleotides containing siRNA construct at a length of 63bp with 5'-terminal phosphorylation were chemically synthesized respectively. The complementary DNA strains were annealed and inserted into the downstream of U6 promotor of the pSilencer 2.1-U6 neo vector to construct siRNA expression vectors pSi-RdRp1286, pSi-RdRp1441 and pSi-OCP117. PCR and sequencing were carried out to identify the expression plasmids. The result showed that expression plasmid pSi-RdRp1286, pSi-RdRp1441 and pSi-OCP117 were successfully constructed.