| Grass carp reovirus(GCRV) is the strongest virulence of aquatic reovirus and the most harmful virus in China’s freshwater aquaculture. Grass carp hemorrhage caused by the virus, causing huge economic losses to our grass carp aquaculture each year. Etiology complicated, virus genotype varied and the lack of targeted prevention and control measures cased the disease continuing popularity. So far, there are 30 strains have been reported. There are big difference among strains in the cell culture characteristics, virulence, genome banding pattern, gene sequences and so on.In August 2012, a grass carp farm in the Baiyun district of Guangzhou suspected occurred grass carp hemorrhage diseases. The main clinical symptoms of illness grass carp are severe bleeding intestinal, colitis symptoms, red gill congestion, but other parts have no symptom. Collected intestines, liver, spleen and kidney tissue of diseased fish and cut it into pieces, then grinded to get tissue homogenate to inoculated proboscis snout into fibers cells(PSF) for virus isolation. PSF showed cytopathic effect(CPE) began the second generation with inoculated the virus.Electron microscopy sections prepared by negative staining method. Virus particles are hexagonal, no envelope, about 70 nm and was lattice-like arrangement. At the same time detected these tissues and the cell by triple RT-PCR method and amplified about 500 bp specific band, the gut of that is the most obvious. The product which result is positive were sequenced.Compared the sequence with corresponding sequences of 873 and GSRV by BLAST. The similar rate are 93% and 92% respectively. To obtain a high concentration of the virus solution, we cultered PSF cell and infected by the virus. And extracted the viral genome to electrophoresis through SDS-PAGE, the result showed the virus has 11 fragments. Compared with type I 873, their genomes relatively similarity. But there are differences in the position of three large fragments. Combined with clinical symptoms, the result of cell infected with virus, these results of electron microscopy and RT-PCR test, the nucleic acid sequence homology and result of SDS-PAGE, we initially confirmed the isolated virus is grass carp reovirus and tentatively named it as GCRV GZ1208.Obtained the virus titer by determined the TCID50 of the virus, the result is 10-8.5/0.1m L. Infected rare minnow and grass carp with virus solution of 10-3.5/0.1mL. In regression infecyion test and cross-protective immunity test against other genotypes of the virus. The result of infection regression test showed rare minnow and carp after infected the virus the infection rates is 100%, but mortality rates is about 40%. Cross-protective immunity tests showed GZ1208 has no immune protection effect to type II strain HuNan1307, but have a good protective effect to type I strain JX-01.Primers were designed references to genomic sequence of 873. Get the entire gene segments of GZ1208 by RT-PCR amplification and cloning. The full-length genome is 23.696 Kb, the size of S1 to S11 segments are 3949 bp, 3877 bp, 3702 bp, 2320 bp, 2239 bp, 2039 bp, 1414 bp, 1297 bp, 1130 bp, 909 bp, 820 bp. DNAStar,ORF Finder software and GenBank database were used to analysis genomic features, encoding amino acid, homology and phylogenetic tree. The results showed that, in the GZ1208 sequence ends have conserved sequence, similar to the other grass carp reovirus.It is 5’GUUAUUU... UUCAUC3 ’. Nucleotide sequence homologous of segments compared with strain 104(type III) is between 32.4 to 50.6 percent, compared with strain HZ08(type II) is between 38.4 to 52.8 percent, compared with AGCRV is between 52.3 to 69.1 percent, compared with strain 873(type I) is between 90.7 to 98.3 percent, and compared wihe GSRV is between 91.5 to 99.2 percent. The result of phylogenetic tree based on the similarity of the amino acids of each segment showed GZ1208, 873 and GSRV have maximum similarity, clustered into the same branch. The result of sequence homology show that in some segment,the sequence homology with 873 are higher than that with GSRV, while in others,the sequence homology with GSRV are higher than that with 873. These results showed that the virus could be formed by different strains by reconfiguring.These results show that the newly isolated strains GZ1208 belong to type I GCRV. There are similar in genome belt type and characteristic compare with GSRV and the representative strains of type I 873. Several segments of GZ1208 and 873 share a higher similarity than that of GSRV, while some other segments of GZ1208 and GSRV share a higher similarity than that of 873. Concluded that the newly isolated strain may be reconstituted by other different strains. The new strain not only enrich the grass carp reovirus database and genome sequence database, but also laid the foundation for the study of genetic variation regularity of GCRV. |
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