The Influence On IHNV Virulence And Immunogenicity With Mutation Of G Protein Potential N-glycosylation Sites

IHNV belongs to the genus Novirhabdovirus,family Rhabdoviridae,and produces contagious acute infection in juvenile salmonid fish,resulting in hemorrhage and necrosis of hematopoietic tissue and other internal organs.IHNV is widely prevalent in North America,Europe and Asia and has become a worldwide rainbow trout disease,causing great economic losses in rainbow trout breeding.Thus far,there is no effective treatment for IHNV.Early vaccination in healthy fish is an effective method to prevent viral infection.Although there are many reports of the IHNV vaccine,only one type of DNA,"vaccine APEX-IHN",has acquired vaccine production licenses in Canada.IHNV is a nonsegmented negative-strand RNA virus with a genome length of approximately 11100 bp..The complete genome sequence of IHNV contained six genes that encode five structural proteins and one non-structural protein.They are the nucleocapsid protein(N),the phosphate protein(P),the matrix protein(M),surface glycoprotein(G),the non-virion(NV)protein,and the polymerase protein(L).Among of them,G protein is not only glycoprotein,but also the virulent protein,and contains the main antigenic determinant,which can stimulate organism producing neutralizing antibody,and play a vital role in the pathogenicity of IHNV and the immune response.The glycosylation of outer membrane protein is a general phenomenon in the processing of virus reproduction.The Mutation of glycosylation sites can affect the folding and conformation of virus protein,virus reproduction and immunogenicity in organism.In order to study the influence for mutation of G protein N-glycosylation sites on IHNV virulence and immunogenicity,in our study,four protential N-glycosylation sites of IHNV G protein were forecasted on line.The amino acids of 56th,379th,401th and 438th were changed(Asp to Ala)with single point mutation,three points mutation and four points mutation.These mutations were identified by digestion and sequencing.Nine recombinant viruses were rescued by reverse genetic system,which can generate CHSE-214 cells CPE.Nine recombinant viruses were confirmed by RT-PCR,genetic tags,indirect immunofluorescence assay(IFA)and electron microscope observation.The glycosylation level was analyzed by Western Blot,and comfirmed that the four sites were wttIHNV HLJ-09 G protein N-glycosylation sites.Mutating the amino acid of glycosylation sites can be deglycosylation.The growth culves showed that the graphetic level of rIHNV-N56A-N401 A-N438A、rIHNV-N379A-N401A-N438A and rIHNV-N56A-N379A-N401A-N438A were significantly lower than the other recombinant viruses and the parent virus,which illustrated when the 401th and 438th amino acids of G protein were mutated at the same time,the graphetic level can obviously decreased.In the cell tropism experiment,the replication level of the nine recombinant viruses were the highest in CHSE-214 and the lowest in RTG-2 as same as the parent virus.To assess the pathogenicity and virulence level of the recombinant viruses,the juvenile rainbow trout of lg were challenged by intraperitoneal injection with these.The results showed that in 30d,the cumulative mortality of the group of rIHNV-N438A was below 50%only,which can illustrated the mutation of 438th amino acid of G protein can reduce the cumulative mortality of juvenile rainbow trout.Fish challenged with recombinant viruses exhibited clinical signs typical in liver,kidney and heart.The viral copy numbers in liver and kidney of fish challenged with recombinant viruses was detected with the method of real-time PCR,and the result showed the groups of rIHNV-N56A-N401A-N438A、rlH’NV-N379A-N401A-N438A and rIHNV-N56A-N379A-N401A-N438A were the lowest,which testified when the 401th and 438th amino acids of G protein were mutated at the same time,the graphetic level in vivo can also obviously decreased.Indirect ELISA result showed that high level serum antibody was detected after challenged with recombinant viruses.This indicated that rIHNV could induce system immune responses in the experimental fish.Among them,the specific antibody lgM can be produced earlier in the group of rIHNV-N438A,inferring that the mutation of 438th amino acid of of G protein may relate to "immune escape.".The surviving fish of rIHNV-N438A group were challenged with the parent virus again,and the the cumulative mortality was 25%in 30d,indicating that the fish challenged with rIHNV-N438A can resist IHNV.The study comfirmed that the glycosyl makes important effects on virus,which provide a basis for the pathopoiesis mechanism and immunomechanism of IHNV,and the attenuated live vaccine against IHN.