Research On The Prevention Of IHN And IPN With A Bivalent Attenuated Vaccine

Infectious hematopoietic necrosis(IHN)and infectious pancreatic necrosis(IPN)are acute,highly contagious systemic diseases that pose a serious threat to salmon and trout aquaculture.The outbreak of these two diseases caused huge economic losses to worldwide salmon and trout industry.The pathogen for IHN is the infectious hematopoietic necrosis virus(IHNV),which belongs to the Rhabdoviridae family,Novirhabdovirus genus,and the Salmonid novirhabdovirus species.The virus genome is an unsegmented negative-sense single-stranded RNA.The pathogen for IPN is the infectious pancreatic necrosis virus(IPNV),which belongs to the Birnaviridae family and Aquabirnavirus genus.The virus genome is a two segmented double-stranded RNA.For IHN and IPN,although there are currently commercially available DNA vaccines and inactivated vaccines respectively,due to import restrictions and biosafety issues,there is still no commercial vaccine in China,let alone a dual vaccine against the above two diseases.Therefore,this study will carry out the research on the dual vaccine against IHNV and IPNV.The establishment of reverse genetic system provides a technical platform for the study of virus gene function,pathogenic mechanism,and novel virus vector vaccines.In order to study the attenuated vaccine for IHN and IPN,this study will determine the endemic strains of IHNV through viral phylogenetic analysis and transcriptome analysis,and establish the reverse genetic operating system of IHNV to determine the optimal site for recombinant IHNV virus to express exogenous proteins as the carrier.Finally,the recombinant virus that can successfully express IPNV VP2 protein was constructed,and the recombinant virus was used as a vaccine to protect rainbow trout against IHNV and IPNV infection.The following is the main content of this research:1.Through evolutionary analysis of the system,it has been found that the currently prevalent IHNV strains in China belong to the J genotype,which is highly pathogenic in rainbow trout.Transcriptome analysis of host responses to IHNV infection with U and J genotypes revealed significant differences in host immune responses between the two strains.2.The G protein of IHNV and the VP2 protein of IPNV were expressed and purified using prokaryotic expression systems.Polyclonal antibodies against IHNV and IPNV were prepared using these two proteins,respectively,and were applied in the evaluation of a bivalent attenuated vaccine in this study.3.In this study,a plasmid p IHNV-Sn1203 was successfully constructed,which expressed the full-length c DNA of the virus,as well as auxiliary plasmids expressing the N,P,Nv,and L proteins of the virus.After co-transfection with BHK-21 cells expressing T7 polymerase,a viable recombinant virus rIHNV-Sn1203 was successfully rescued.Multiple biological characteristic identification assays were performed on the recombinant virus,which showed that the recombinant virus maintained similar biological characteristics to the wild-type virus,indicating the successful establishment of the IHNV reverse genetic system.4.To determine the optimal expression site of exogenous genes in IHNV as a vector,this study inserted the GFP gene as an exogenous gene between different IHNV viral genes.By measuring the fluorescence intensity of GFP protein,it was found that the highest expression level of GFP protein was in rIHNV-GFP-P/M,followed by rIHNV-GFP-N/P,rIHNV-GFP-M/G,rIHNV-GFP-G/Nv,and rIHNV-GFP-Nv/L.These results indicate that the P/M gene region is the optimal expression site for exogenous genes in IHNV as a vector.5.In order to develop a bivalent vaccine against both IHN and IPN,the attenuated IHNV strain Blk94(for rainbow trout)was used as a vector and VP2 gene of IPNV virus was inserted into P/M gene region.The protective effect of this attenuated vaccine found that the relative survival rate of rainbow trout after infection with IHNV was 86%,and the viral load of IPNV in the tissues was significantly reduced.The immune-related genes were significantly upregulated.At the same time,after immunization with this attenuated vaccine,rainbow trout produced high levels of neutralizing antibodies against both IHNV and IPNV.Conclusion: The currently prevalent IHNV strains in China belong to the J genotype.Polyclonal antibodies against the IHNV G protein and the IPNV VP2 protein have been successfully prepared.An IHNV reverse genetic system has been established,and the optimal location for expressing exogenous genes as IHNV carriers between the P/M genes has been determined.A bivalent attenuated vaccine has been successfully developed for the prevention of IHN and IPN,which lays a solid foundation for the development of a live attenuated vaccine for rainbow trout.