Development Of Live Vector Vaccine For Infectious Hematopoietic Necrosis

Infectious hematopoietic necrosis(IHN)which was caused by Infectious hematopoietic necrosis,is a highly contagious disease that can cause to hematopoietic necrosis with kidney and spleen as the main feature on salmon trophies.The pathogen of the disease is infectious hematopoietic necrosis virus(IHNV),is a negative-strand RNA virus,belonging to the rhabdovirus Corneloid virus genus.IHN is mainly against fry and species of fish,the incidence of fish mortality is usually more than percent 90,is the first category of fish port quarantine objects,by our country as a second animal disease.IHN has caused devastating blows to salmon trout farming in countries such as North America,Europe and Asia,which has seriously obstructed the development of aquaculture in various countries.Outbreak of IHN on China in 1990 in Liaoning,the mortality rate of nearly percent 100.In recent years,with the rapid development of Chinese industry of rainbow trout,in-depth study of IHNV for better prevention and treatment of the disease has important practical influence.In this study,we established isolation and identification of IHNV,and cloned G gene of IHNV,and the adenovirus was used transfer vector of exogenous gene to construct recombinant adenovirus vector expressing G protein of IHNV.The study with safety test,the best use of dose determination test,efficacy test,immune duration and shelf life of recombinant adenovirus with G gene of IHNV and IHN live vector vaccine,laid a solid basis for infectious hematopoietic organ necrosis better prevention and control for the next step,and it was named IHNV GS strain.1.To isolate and identify pathogen of suspected hematopoietic organ necrosis(IHN)in the rainbow trout farm in Gansu Province.The N gene was isolated from the appropriate cell lines(RTG-2 cells,CHSE-214 cells and EPC cells)and amplified by RT-PCR and PCR.The N gene was cloned into the vector with p MD-19 T,and transformed into E.coli to identify the virus by PCR amplification.The results showed The typical cytopathic effect(CPE)was appeared by that the rainbow trout gonadal cells(RTG-2),the larvae of the larvae(CHSE-214)and the carp epithelium cells(EPC)were inoculated with the suspensions were grinded by samples.The virus was used to amplify with the N gene,The N gene of IHNV was cloned successfully and the length was 786 bp.And the homology of nucleotide sequences of the N gene of LB91 KI strain,RB76 strain,HV-7601 strain and HLJ-09 strain,which were published as representative strains of IHNV,were 99%,99% and 97% respectively.And 96%.The deduced amino acid homology was 99%,97%,96% and 98%,respectively.The phylogenetic tree was constructed by using MEGA software.The phylogenetic tree analysis showed that the isolates were close to the American strain JB91 KI,RB76,Chinese strain HLJ-09 and Japanese strain strain HV7601.The homology of the deduced amino acids was respectively 99%,97.8%,96.5% and 98%.The results show that the outbreak of the disease is infectious hematopoietic necrosis,was caused with infectious hematopoietic organ necrosis virus.2.To clone G gene of infectious hematopoietic organ necrosis(IHNV),and to construct recombinant adenovirus vector of infectious hematopoietic organ necrosis(IHN)with G gene,and to provide reference for the prevention of IHN and the development of vaccine.The G gene was amplified by RT-PCR and PCR.And was inserted into the adenovirus shuttle vector.The adenovirus vector with the target gene was recombined with the backbone vector in Escherichia coli BJ5183 to construct the recombinant plasmid.The recombinant plasmid was transfected into HEK-293 cells after digested with Pac I.The recombinant adenovirus with the target gene was identified by PCR amplification and digestion with sal ? and xho ?.The recombinant adenovirus was monitored by the green fluorescence protein and was detected expression of glycoprotein by Western-blot.And the recombinant adenovirus was determined by TCID50.The G gene of IHNV was cloned successfully and the length was 1533 bp.And the homology of the isolated IHNV GS strain was 99% with the strains IHNV Ch YU78,Ch Ab76,Ko Mo71,Rt Naq82 and HV7601 strains.Phylogenetic tree analysis showed that the IHNV isolates were close to the Korean strains CHYU78,Ch Ab76,the American strains Auke77 and Blk94.The result that amplification of PCR and digestion of restriction enzyme showed that recombinant adenovirus with IHNV G protein was successfully constructed.Monitoring of GFP showed that recombinant adenovirus was transfected successfully.The analysis of Western blot showed that recombinant adenovirus could express about product of 58 ku in HEK-293 cells,which was consistent with the molecular mass of anticipation.The assay with TCID50 of recombinant adenoviruses showed that the titer of recombinant adenovirus reached 1.0×1010.4/m L.It showed that having been successfully constructed with infectious hematopoietic organ necrosis(IHN)G gene recombinant adenovirus vector,and recombinant adenovirus has stable and high titers.3.To complete the live vector vaccine of IHN and evaluate the effect of the vaccine.With the safety test,the best use of dose determination test,efficacy test,immunological duration and shelf life test operation of live vector vaccines of IHN.The results showed that there was no symptoms of swollen,systemic reactions and infectious hematopoietic necrosis in adult guinea pigs,adult mice and rainbow trout.Immunological rainbow trout was tested according to different doses.The results showed that the pool was infected with IHN,The survival rate of 0.1 m L immunized fish was the highest.Different immunological factors in the rainbow trout after immunization were determined with q RT-PCR.And the immune factors of the rainbow trout were significantly increased after immunization compared with the control group.The antibody level of the serum of the rainbow trout was measured,and gradually become positive(NI> 50)and continued to increase compared with the control group.And attacking by IHNV with the immune rainbow trout.The results showed that the vaccine had a good protective effect on the rainbow trout after immunization.Immune juveniles after soaking for two weeks were observed in the infected pool with IHNV,the growth of rainbow trout was good within 6 months,sporadic death occurred 6 months later,and the typical symptoms of IHN were found.PCR products were positive by PCR amplification.In a word,the immunization duration of the live vector vaccine of IHN is tentatively scheduled for 6 months.In the case of preservation at 2-4 °C,the survival rate of the immunized fish and the data of the efficacy test were close to each other,so the shelf life was tentatively set at 12 months.The results showed that the live vector vaccine of IHN was safe and effective,and the use method was simple.The actual dosage of each fish is 0.1 m L.The duration of the immunization was 6 months and the shelf life of the vaccine was tentatively scheduled for 12 months.In summary,we established the method of isolation and identification of IHNV,constructed the recombinant adenovirus vector with IHNV G gene,and completed the development of live vector vaccine of IHN.With the safety test,the best use of dose determination test,efficacy test,immunological duration and shelf life test operation of live vector vaccine of IHN.It laid a solid foundation for further better prevention and control on infectious hematopoietic necrosis.