| Infectious hematopoietic necrosis virus(IHNV)belong to the Rhabdoviridae,Novirhabdovirus,and the virus can cause an acutes,deadly,hemorrhage and necrosis disease of hematopoietic tissue and other internal organ of young salmon diseases.Infectious hematopoietic necrosis(IHN)is a kind of important viral diseases that can be harmful to salmon industry and has strong pathogenicity and highly contagious.The disease has different fatality rate according to the size and type of fish,different strains and environmental factors,which can reach 100% when it is serious and it cause huge economic losses in aquaculture industry all over the world.Suspected cases of IHN in rainbow trout farms in Yongdeng county of Lanzhou city of Gansu province caused the partly mortality of rainbow trout.After sampling,experiments were conducted as following:(1)The laboratory identification of the disease was conducted which contains isolation of IHNV,RT-PCR identification,homology and genetic analysis.The result showed that typical cytopathic effect(CPE)can be seen in endothelial progenitor cell(EPC)which was exposed in suspension of pathologic materials that were grinded and filtered.A 1,408 bp fragment was obtained after propagating the N gene of this virus.The homology comparison and analysis of the phylogenetic tree for this fragment showed that the nucleotide sequences homology of isolate with the known Ch2010008?HLJ-09 ? CJ-13 ? IHNV-PRT strain was 99.7% ? 99.7% ? 99.2% and 93.7% respectively,and amino acid homology of isolate with the Ch2010008?HLJ-09?CJ-13?IHNV-PRT strain was 100%?100%?99.0% and 89.3% respectively.Polygenetic tree showed that isolate gathered together with Chinese strains of Ch2010008?HLJ-09?CJ-13 and Korean strain of IHNV-PRT,which means a close genetic relationship.The isolate was named GS1,and it is the cause of these cases.(2)In order to further study the biological characteristics,origin and propagation path of IHNV GS1,10 pairs of specific primers were designed to amplify the genome of virus,and 3 pairs of specific primers were designed for 3',5' race.Homology analysis and phylogenetic tree analysis of nucleotide and amino acid sequences of 6 structural proteins were conducted.The result showed: the length of IHNV GS1 sequence is 11,134 bp,and the Phylogenetic tree analysis showed N,P and M gene of IHNV GS1 is most closely related South Korea strain of PRT IHNV,and G genetic strain is most closely related South Korea Rt Gu01 strains,Rt Ui02,Ch Ya07 and Japan Rt Nag96.The results show that the IHNV GS1 belongs to the JRt genotype,the strain may originate from South Korea or Japan,and the virus may spread by trade of IHNV contaminated fish eggs,larvae and young fish.(3)Up to now,none of vaccine can be used for IHNV except for one kind of DNA vaccine made in Canada can be usd in Canada.Formaldehyde and ?-propiolactone(BPL)were used for develop inactivated vaccine of IHNV GS1.After the physical properties testing,detection of residual amount of virus,sterility testing,safety examination,shelf life test and potency test,pink or milk white powder of inactivated vaccine can be obtained.Pipette(1 m L)with inner diameter of 1.2 mm was used to draw 1m L vaccine at room temperature,and 0.4 m L of vaccine can flow naturally in 0.4 s of time.Standby assumes the shape of drops onto the surface of cold distilled water,it can be observed that two kinds of vaccines are not spread,which means the vaccine was water-in-oil dosage forms.Two kinds of inactivating agent inactivated antigen inoculation carp EPC,and blind passage for 2 times,the cell were observed under inverted microscope,it is found that cell were free from lesions.After the inactivated antigen were injected to rainbow trout,no rainbow trout death phenomenon happened.Inactivated antigen inoculation of ordinary,anaerobic meat,liver soup broth medium media,ordinary AGAR medium and blood AGAR medium,none of medium turbidity occurred and cultures are of colonies appears.After two kinds of inactivating agent of inactivated vaccine were injected to rainbow trout for 30 days,none of rainbow trout,local swelling,systemic reactions means the vaccine is qualified;Vaccine effectiveness experiment showed that two kinds of inactivating agent inactivated vaccine have better protection efficacy.Preliminary vaccine after 12 months storage under 4 ?were injected to rainbow trout,the survival rate close to effectiveness testing,which means the tentative retention period for 12 months.Two kinds of inactivated vaccine have better protection efficacy,which may be helpful to further development of the IHNV inactivated vaccines. |
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