Development Of A Combined Live Vector Vaccine Against Infectious Hematopoietic Necrosis And Infectious Pancreatic Necrosis In Rainbow Trout

Infectious hematopoietic necrosis(IHN)which was mainly caused by infectious hematopoietic necrosis virus(IHNV),is a high mortality rate affecting mainly salmonid fish that can cause to the main feature with hematopoietic necrosis of kidney and spleen on natural and artificial conditions.IHNV is a negative-strand RNA virus belonging to the genus Rhabdovirus.Infectious pancreatic necrosis(IPN)is the etiological agent of infectious pancreatic necrosis virus(IPNV),which causes serious economic losses for salmonid aquaculture.IPNV is a non-enveloped,double-stranded RNA virus(segments A and B)and the type species of genus Aquabirnavirus of the family Birnaviridae.IPNV is the smallest RNA virus among currently known fish virus diseases.Both diseases mainly cause young fish,larvae and fry of salmonids,with a mortality rate of over 90%.IHN was fixed class 1quarantine objects at aquatic animal ports and was listed as the class 2 animal disease by the Ministry of Agriculture and Rural Affairs.In China,IHN and IPN were first detected in northeastern area in 1990 and 1986,respectively.Both diseases have brought a heavy blow for the farming of salmon and trout all over the world,which has seriously affected the aquaculture industry in various countries.1.Carp epithelial tumor cells(EPC)were used for cell passage of IHNV and IPNV,respectively.The virus was passed at 14°C.When the cells were CPE with the virus,the temperature began to rise to 18°C.Pass the collected virus on normal EPC cells.In this study,a strain of IHNV was successfully domesticated,and stable CPE was appeared within 48hours at 16-18?.It named by IHNV GS strain.At the same time,a strain of IPNV was domesticated,and stable CPE was appeared within 48-60 hours at 18-20?.The IHNV GS strain and IPNV GS strain were amplified by the conservative gene using PCR method,respectively.And the diagnosis method was established for the two viruses with the molecular level.The suspected outbreak of IHNV in Yongdeng County and outbreak of IPNV Yongjing County,Gansu Province were confirmed respectively.The IHNV G gene and IPNV VP2 gene were analyzed by the phylogenetic tree,respectively.The results showed that the IHNV G gene and the representative strains of IHNV has a relatively close genetic relationship such as IHNV Ch YU78,IHNV Auke77 and IHNV Ch Ab76.The IPNV VP2 gene has a relatively close genetic relationship with the representative IPNV strains such as IPNV SP,IPNV Denizli05,IPNV HAH-3 and IPNV Ka470/07.The molecular diagnostic methods were established for IHNV and IPNV,and the method which cultures virus through cell lines in salmon and trout in this experiment,provide a theoretical basis for better clinical diagnosis of IHNV and IPNV.2.To clone infectious hematopoietic necrosis virus(IHNV)G gene,F2A linker gene and and VP2 gene of infectious pancreatic necrosis virus(IHNV),respectively.And construct recombinant adenovirus vector of IHNV G gene,F2A gene and IPNV VP2 gene.The IHNV G gene,F2A gene and IPNV VP2 gene were amplified respectively by PCR method,and which were inserted to the shuttle vector using multi-segment quick fusion technology.Linearized recombinant vector plasmids and the backbone vector were recombined with a recombinant adenovirus plasmid in Escherichia coli.After PCR amplification and enzyme digestion with Not?and Hind?,the plasmid was transfected into 293 cells for virus packaging after Pac?digestion.To obtain the recombinant adenovirus with target gene with monitoring the recombinant adenovirus by observing the green fluorescence pretein(GFP).The expression of G protein and VP2 protein were detected by Western-blot.And the titer of the recombinant adenovirus was detected.The results showed that IHNV G gene,F2A gene and IPNV VP2 gene were successfully cloned.And the total length of the gene was 3036 bp.The recombinant adenovirus of IHNV G,F2A gene and IPNV VP2 gene was successfully constructed.GFP results showed that the recombinant adenovirus was successfully transfected.The recombinant adenovirus had a band of protein at a molecular mass of about58 k D(G protein)and 54 k D(VP2 protein)in 293 cells respectively,which the TCID₅₀reaches 1.0×10^10.0 m L^-1.The results showed that the recombinant adenovirus vector with IHNV G gene and IPNV VP2 gene was successfully constructed,and the recombinant adenovirus had stable and high titer.3.To evaluate the protective effect of live vector vaccine against IHNV and IPNV infection after immunization of rainbow trout.Administered via the immersion route,the live vector vaccine containing the coding region of the IHNV G and IPNV VP2 induced protective immune responses in rainbow trout.q RT-PCR revealed the expression of the IHNV G and the IPNV VP2 in the spleens of vaccinated rainbow trout reached the highest level at 3 days post-vaccination(dpv)and thereafter gradually decreased between 3 and 15 dpv.TLR-3,TLR-7and TLR-8 was upregulated and the highest expression levels of IFN-1,Mx-1,Mx-3,Vig-1and Vig-2 were detected at 3 dpv.Four markers of the adaptive immune response(CD4,CD8,Ig M and Ig T)were continuously increased in the spleens of vaccinated fish as compared with controls,between 3 and 15 dpv.For best protection,juvenile rainbow trout should be immunized with a 1:100 dilution of the titer with 1×10^10.0 m L^-1 TCID₅₀ of the live vector vaccine,via 10-min immersion.The cumulative percentage mortality differed significantly between the vaccinated fish and the controls(empty-plasmid-vaccinated).Antibody detection revealed vaccinated rainbow trout displayed high levels of serum antibodies against IHNV infection and IPNV infection.And the relative percent survival value(RPS)reached 87.50%after IHNV challenge,and the relative percent survival value(RPS)reached 86.84%after IPNV challenge.Taken together,our results demonstrate that this is a promising live vector vaccine that could be used to protect rainbow trout against IHNV infection and IPNV infection.In summary,this methods for cell line diagnosis and molecular diagnosis of IHNV and IPNV has been established,respectively,and recombinant adenoviruses with IHNV G gene and IPNV VP2 gene was constructed.The live vector vaccine was evaluated protective effect against IHNV infection and IPNV infection in rainbow trout,respectively.It provides a reference for prevention and control of infectious hematopoietic necrosis and infectious pancreatic necrosis.