Construction And Safety Assessment Of A DNA Vaccine Against Infectious Hematopoietic Necrosis(IHN)

Infectious hematopoietic necrosis virus(IHNV),belonging to the family Novirhabdovirus within genus Rhabdoviridae,is the pathogen of infectious hematopoietic necrosis disease(IHN).IHN is one of the most important viral diseases of salmon and trout.The outbreak of IHN will result in a 100% mortality rate of salmon trout fry,which has caused huge economic losses to the world's salmon and trout farming industry and seriously hampered the healthy development of salmon trout farming.Since introduced to China in 1980 s,no effective drug was developed against IHN.No commercial IHN vaccine can be directly introduced to China due to viral mutation and safety concerns.This study describes the development of a nucleic acid vaccine against IHN,the protection efficacy assessment and kinetic distribution of the DNA vaccine in the inoculation sites in rainbow trout.Furthermore,the effluence of the ampicillin resistance gene from the DNA vaccine to in vivo and in vitro environmental microbes was evaluated in this study.In this study,the glycoprotein(Glycoprotein,G)gene from the IHNV isolate SD-12 was cloned into the eukaryotic expression vector pc DNA3.1(+)to construct a nucleic acid vaccine against IHN(designed p IHNsd-G).Rainbow trout(Oncorhynchus mykiss)fry(5 ± 0.5 g)was immunized with a 2 ?g of the nucleic acid vaccine using intramuscular injection at the dorsal fin base.At the 4 days post vaccination(d.p.v.)and the 30 d.p.v.,the protection rate was measured by intraperitoneal injection of IHNV isolate SD-12 at a dose of 100 –fold half tissue culture infection dose(TCID50).The expression of Mx-1 gene in the kidney and inoculated muscle of the immunized rainbow trout was measured by real-time PCR at the 4 and 7 d.p.v.IHNV neutralizing antibody titers were determined at 60 and 150 d.p.v.During the 65 days after immunization,the contents of feces,water and intestinal contents of the rainbow trout were collected at different time points to isolate and identify the ampicillin resistant bacteria.At different time points post-vaccination,the promoter sequence of the nucleic acid vaccine vectorand the ampicillin resistance gene sequence were used as the target gene,and the dynamic distribution of the nucleic acid vaccine in the inoculated muscle of ??the immunized rainbow trout was monitored by PCR method.The relative protection rates of the nucleic acid vaccine were 93.4% and 91.4% at 4and 30 d.p.v.,respectively,and the protection rate of the p IHNsd-G on rainbow trout could reach more than 89% at 60 and 150 d.p.v.The results suggest that the nucleic acid vaccine p IHNsd-G has a good protective effect on rainbow trout and is resistant to attack of IHNV on rainbow trout.The results of Mx-1 gene expression analysis showed that the Mx-1 gene expression the head kidney and the inoculated muscle were significantly upregulated compared with the PBS control group at 4 and 7 d.p.v.(P<0.01).This result provided a reasonable explanation for the early nonspecific protection of the IHN nucleic acid vaccine.The titration of the neutralizing antibody showed that no neutralizing antibody was present in all sera at 4 d.p.v.(titer <20),and neutralizing antibodies were determined from 8 out of 10 fish sera at 30 d.p.v.,and the highest titer was as high as 160.Neutralizing antibodies were detected in all immunized rainbow trout serum,and the highest titer was 320 at 60 d.p.v.At 150 d.p.v.,neutralizing antibodies were detected in nine out of ten fish serum,and the highest titer was 80.The results indicated that p IHNsd-G was able to stimulate the rainbow trout specific immune responses,which enabled the immune rainbow trout to produce antibodies to neutralize IHNV attack.The results of ampicillin resistant bacteria isolation showed that the isolated ampicillin-resistant bacteria belonged to Aeromonas sp.and Aerrobacter sp.,which were naturally resistant to ampicillin,and no Escherichia coli was isolated.No significant change in the species or number of ampicillin-resistant bacteria was observed between the experimental and control groups,demonstrating that the resistance gene of the nucleic acid vaccine was safe.The PCR results of p IHNsd-G distribution showed that all p IHNsd-G target fragments were detected in the muscle of the injection site at 1 d.p.v.,full-length ampicillin resistance gene could not be amplified from the injection site muscle at 84 d.p.v.,and all target genes disappeared at 150 d.p.v.This study not only provided a high efficient nucleic acid vaccine against IHN,but also laid the foundation for the safety evaluation of the nucleic acid vaccine.