Isolation And Identification Of Red-Spotted Grouper Nervous Necrosis Virus And The Mechanism Of B2 Protein Inhibiting The RLRs-Mediated Type ? Interferon Response

Viral nervous necrosis(VNN),whose pathogen is nerve necrosis virus(NNV),is one of the major viral threats to worldwide aquaculture.So far,it has been reported that more than 120 kinds of fish,including marine and freshwater fish,could be infected by NNV around the world in addition to South America.In serious cases,NNV infection can cause larval and adolescent mortality as high as 100%,resulting in huge economic losses worldwide.In the present study,we isolated a new RGNNV strain named RGNNV-HL strain from cultured adult hybrid Hulong grouper(Epinephelus fuscoguttatus(?)× Epinephelus lanceolatus(?))in Guangdong province and three monoclonal antibodies(MAbs)against the coat protein of red-spotted grouper nervous necrosis virus(RGNNV)were produced,then the mechanism which B2 inhibits host RLRs-mediated type ? IFN response was investigated.Findings from this study will provide a candidate material and a useful epidemiology information for further study on the infection mechanism and prevention and control strategies for RGNNV.The detailed contents and results are as follows:(1)Isolation and identification of RGNNV from adult hybrid hulong grouper(Epinephelus fuscoguttatus × Epinephelus lanceolatus).The virus was identified by cell culture,RT-PCR,tissue section,electron microscopy and whole genome sequencing.Artificial infection test for virulence test were carried out.General examination of affected fish showed no noticeable lesion,no parasite in the gills or body surface,and no pathogenic bacterium.Pathological examination showed that there were serious vacuoles in the brain tissue of the diseased grouper.SSN-1,GF-1and GS cells showed obvious cytopathic changes after inoculating with the homogenate of diseased fish.Electron microscopy showed that there were icosahedral virus particles about 30 nm in size,similar to betanodaviruses,in infected-GS cells.RT-PCR and sequencing analysis confirmed the existence of the capsid protein sequence of RGNNV.The virus was clustered with RGNNV genotype on the phylogenetic tree based on RNA1 and RNA2 sequences.Therefore,the new virus isolated from adult Hulong grouper is tentatively named RGNNV-HL.Artificial infection experiments showed that experimented fish began to die from 4th and 5th day after intraperitoneal injection and immersion.The cumulative mortality of injection and bathing was 93.3% and 80% respectively.(2)Generation and characterization of monoclonal antibodies(MAbs)against the coat protein of RGNNV.The recombinantly expressed Cp of RGNNV was used as the immunogen to prepare MAbs using hybridoma cell fusion technology.Three MAbs were obtained by indirect enzyme-linked immunosorbent assay(ELISA).Western blotting results show that the three MAbs can specifically recognize the Cp of RGNNV.The result of IFA showed that the MAbs could recognize virions in RGNNV-infected grouper spleen(GS)cells.These results indicated that the MAbs could recognize RGNNV virions and could be used to produce a rapid detection method.This study provides a foundation for further studies on the rapid diagnosis of RGNNV and its infection mechanisms.(3)RGNNV B2 protein inhibited fish interferon production by suppressing cell transcription guided by RNA polymerase ?.We firstly found that the upregulation of IFN?1 promoter activity stimulated by poly(I:C)was suppressed by RGNNV infection.By systematically screening the structural and nonstructural proteins of RGNNV,we identified B2 protein as the virus' s most potent antihost protein,which inhibited the IFN?1 promoter activities stimulated by MDA5,MAVS,TBK1,IRF3,and IRF7 and decrease their protein expression.Importantly,B2 also suppressed the constitutively active cytomegalovirus(CMV)promoter,which indicated that B2 might be a general inhibitor of gene transcription.Moreover,western blotting showed that B2 could decrease RNAP ? expression and RNAP ? phosphorylation.All these results showed RGNNV blocked host's IFN response by B2 protein directly dysregulating RNAP ? expression.As we know,the study is the first report about the mechanism of RGNNV for anti-host IFN response.These results will provide a foundation for further research on the infection mechanism and effective control strategies of RGNNV.