Isolation and identification of channel catfish (Ictalurus punctatus) hemorrhage reovirus / 病毒学报

By using cell culture and virus infection methods, a new reovirus had been isolated from channel catfish (Ictalurus punctatus) suffered with severe hemorrhage and had been identified as channel catfish reovirus (CCRV) after artificial infection in fish, electron microscopy observation, physical-chemical tests, genomic SDS-PAGE analysis and sequencing. In artificial infection test, the typical symptoms of channel catfish hemorrhage as naturally occurred could be reproduced. The isolated virus could cause typical cytopathic effect in CCO and CCK cell lines. Electron microscopy observation of ultra-thin section samples of CCRV infected CCO and CCK cells revealed that the virus replicated in cytoplasm, arrayed in crystalline, and had a non-enveloped double capsid with a diameter of 60-70 nm. Frozen-thawed, 56 degrees C 1 h, chloroform and ether had no significant effects on CCRV titer, 65 degrees C 1 h could significantly inactivated the viral infectivity. The CCRV genome SDS-PAGE analysis and nuclease sensitivity test showed that the virus genome was the same as that of viruses in Aquareovirudae and consisted of 11 segments of dsRNA assigned into three classes L1, L2, L3; M1, M2, M3 and S1, S2, S3, S4, S5 with a range of size from 0.9 to 4.4 kb. The Cloning and sequencing of the CCRV S4 segment indicated the nucleic acid number of CCRV S4 was 909 bp in length, which was exactly the same as that of GCRV S4 (AF403396) and GSRV S4 (AF403407) segments. The BLAST of CCRV S4 sequence in NCBI GenBank showed that it had a 99% and 90% similarity in sequence to the GCRV S4 and GSRV S4 segments, respectively.

Highly efficient inhibition on replication of grass carp reovirus mediated by chemically synthesized small interfering RNAs / 病毒学报

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).