The Construction Of Reverse Genetic System Of Nervous Necrosis Virus And Virus Modification

The nervous necrosis virus(NNV)belongs to the family Betanodavirus.It causes viral nervous necrosis(VNN)in fishes,which mainly attacks the central nervous system of fishes and causes cell vacuolation lesions in the infected parts.Nervous necrosis virus causes high morbidity and mortality rates in both juvenile and adult fish worldwide,greatly affecting the development of marine and freshwater aquaculture.NNV is an unenveloped single-stranded,two-segment RNA virus,it contains genomic RNA1 and RNA2 and transcriptional subgenomic RNA3,encoding RNA-dependent RNA polymerase,capsid protein and B2 protein respectively.At present,the prevention of nervous necrosis virus infection was focused on mainly in the market,and it carries out disinfection at both horizontal and vertical levels to control the spread and infection of the virus.Now there are few studies on the vaccine of nervous necrosis virus,and the main vaccines are inactivated vaccines and subunit vaccines constructed by capsid proteins,which have some disadvantages such as insufficient protection and adjuvant.Therefore,this project intends to conduct attenuated modification of nervous necrosis virus to provide a new way of thinking for subsequent vaccine research.1?A set of reverse genetic operating system was first constructed for the rescue of nervous necrosis virus in vitro,so as to facilitate the subsequent genetic manipulation experiments.In this study,four different rescue schemes based on three sets of NNV packaged plasmids with different promoter strategies(B7GG cells were transfected with T7 prokaryotic promoter and co-cultured with SSN-1 cells,293 T cells were transfected with CMV eukaryotic promoter,293 T cells were mixed with B7 GG cells transfected with T7 prokaryotic promoter,and B7 GG cells were transfected with T7 prokaryotic promoter)were explored.Finally,a rescue system suitable for nervous necrosis virus was established successfully(B7GG cells were transfected with T7 prokaryotic promoter).2?The nervous necrosis virus B2 protein was mutated,and the NNV virus attenuated strain was constructed by using the established reverse genetic operating system,and the nervous necrosis virus B2 mutant strain was rescued in vitro.The virus proliferation curve of B2 mutant strain was measured.Compared with NNV wild strain,the proliferation rate of unmutant strain and B2 mutant strain was slower,and the virus proliferation ability was decreased.In the fluorescence quantitative-PCR detection of cytotoxic protein Endo-G and antiviral pathway related protein Mx1,the expression of the two proteins caused by rescued unmutated virus and B2 protein mutant virus was far lower than that of wild virus.At the same time,the expression levels of C-reactive protein and tumor necrosis factor caused by B2 mutant virus were lower in vivo than those of wild virus.Meanwhile,the degree of brain hollowing lesion was reduced in vivo in transgenic zebrafish.Therefore,the virulence of the constructed NNV virus B2 mutant is weakened,which is helpful for the development of the genetic engineering vaccine based on B2 mutant virus.3?In addition,the RNA1 segment of the virus was modified according to the high affinity of nervous necrosis virus to the nervous system,and a label with neural loop marking function was added at the 3' end,so as to try to develop nervous necrosis virus into the neuronal loop tracing virus of fish.After using linker(GGGGS)to add e GFP C-terminal short peptide(E11)at the 3' end of RNA1 segment and using reverse genetic operating system for virus rescue in vitro,the virus could not be detected in SSN-1 sensitive cell line infection experiment,RT-PCR detection and e GFP N-terminal(G1-10)transgenic zebrafish in vivo experiment.Again,we attempted to use shorter HA tag and different positions and different link sequences,finally,in the two modification methods of attaching HA tags to the 3' terminal of nervous necrosis virus using the two-fold protein linker(GGGGS GGGGS)and F2 A,we could detect the virus by the expression of viral RNA-dependent RNA polymerase in RT-PCR,the expression of HA tag in cell immunofluorescence assay,and the NNV virus can also be detected in the in vivo injection experiment of transgenic zebrafish.To sum up,we established in vitro rescue method of nervous necrosis virus,and successfully constructed the B2 protein mutant virus,which reduced the toxic effect on the host to some extent.In addition,the method of adding a short peptide of HA to the viral genome with the two-fold protein linker and F2 A was determined,which provides a new method for the study of NNV virus as a neural loop tracer.Due to the limitation of the viral genome and the imperfection of the rescue system,there is a certain gap in the rescue efficiency and proliferation ability of the virus compared with the wild strain,so further exploration and optimization are needed.