The Influence Of NV Protein On Infectious Hematopoietic Necrosis Virus Replication

Infectious hematopoietic necrosis virus(IHNV)is the causative agent of infectious hematopoietic necrosis(IHN),which produces contagious acute infection in a wide variety of salmonid species.IHN causes haemorrhage and necrosis of hematopoietic tissue and other internal organs.Highly virulent strains may cause greater than 90%mortality.This disease is especially destructive in salmonid eggs and fingerlings.Now the disease is widely distributed in North America,Europe and Asian countries,which poses a significant threat to the salmonid farming industry worldwide.Viral infections frequently lead to the activation of host innate immune signaling pathways involved in the defense against invading pathogens.The NF-κB pathway is a central regulator of a host cell’s early response to viral infection.To ensure their survival,viruses have evolved sophisticated mechanisms to overcome the host immune responses.This study was undertaken to determine whether IHNV infection modulates the NF-κB activation in fish cells and to further explore the role of different IHNV viral proteins in modulating innate immune signal transduction.Our results provide a basis for understanding how IHNV affects the NF-κB pathway,which may shed light on the prevention and control of IHNV infection in fish.In this study,we found that infectious hematopoietic necrosis virus(IHNV)infection could activate NF-κB in CHSE-214 cells and EPC cells by using luciferase reporter assay.Then we continued to indentify which IHNV protein contributed to the luciferase activity by using luciferase reporter assay.Our study demonstrated that the IHNV L protein activated NF-κB.However,IHNV NV functioned as an inhibitor of NF-κB activation.An increase in the luciferase activity was higher in the cells overexpressing the L protein compared to the effect of the NV protein on the inhibition of NF-κB activation.In order to determine the mechanism by which NV inhibited NF-κB activity,We compared the subcellular distribution of p65 in control and NV-transfected cells.Cytoplasmic and nuclear fractions of cells were extracted after the cells were treated with SeV and analysed by Western blotting using anti-p65 antibody.The data showed that the level of p65 in the nuclear and phosphorylated p65 were much lower in NV expressing cells,and with the increase of the concentration of NV expressing plasmid,the levels of the phosphorylated p65 and nuclear p65 protein decreased in a dose-dependent manner.The phosphorylation of IκB contributes to the translocation of NF-κB from cytoplasm to the nucleus where of target genes transcription is activated,and IκB degradation in the cytoplasm will occur following its phosphorylation.To get an insight into how NV interfered with NF-κB in the cytoplasm,we examined the effect of NV on the IκBα expression.Overexpression of NV resulted in a relatively high level of IκBα following SeV treatment compared with the control group,which increased in a dose-dependent manner.Evidence gathered to date demonstrated that NV inhibited NF-κB activation characterized by the inhibition of IκBα degradation and p65 nuclear translocation.Real-time qPCR analysis showed that the level of cytokines were lowered by NV due to suppressed NF-κB activation,which suggested that NV attenuated SeV-induced NF-κB activation.To determine the functional domain of the NV protein for NF-κB inhibition,serial deletion mutants of NV protein were constructed,we continued to identify the luciferase activity of mutant NV by using luciferase reporter assay.These data suggested that the amino acid residues of 32EGDL35 responsible for nuclear localization were important for the inhibition of NF-κB activation.We proposed that the region of NV related to the inhibition of NF-κB activation was between amino acids 32-35,therefore,we changed or deleted the amino acids at the corresponding position of wt IHNV HLJ-09 strain.These mutations were confirmed by sequencing and finally a full-length cDNA clone of HLJ-09 mutant strain of IHNV was successfully constructed.Two recombinant viruses were rescued by reverse genetic system.In addition,the two recombinant viruses were confirmed by RT-PCR,genetic tags,indirect immunofluorescence assay(IFA)and electron microscope observation.We tested the impact of recombinant viruses IHNV-NVQ32-35 and rIHNV-NVT32-35 on NF-κB activity in response to TNF-a stimulation,infection with wt IHNV HLJ-09 blocked TNF-a-induced p65 nuclear translocation.In contrast,three recombinant rIHNV-NVQ32-35 and rIHNV-NVT32-35 and rIHNV-ANV-EGFP were unable to inhibit TNF-a-induced p65 nuclear accumulation.The growth curves showed that the replication abilities of the two recombinant rIHNV-NVQ32-35 and rIHNV-NVT32-35 on CHSE-214 cells were significantly lower than that of rIHNV HLJ-09 strain,suggesting that the simultaneous deletion of amino acids 32EGDL35 within the NV significantly reduced the virus replication ability.To assess the pathogenicity and virulence of the recombinant rIHNV-NVQ32-35,rIHNV-NVT32-35 and rIHNV-△NV-EGFP in vivo,juvenile rainbow trout were challenged by intraperitoneal injection with equivalent amounts of recombinant viruses and wt IHNV HLJ-09,and deaths were observed for 20 consecutive days.Fish challenged with wt IHNV HLJ-09 began to die by 4 d,and the peak of death appeared at 6-8 d,While fish challenged with rIHNV NVQ32-35 and rIHNV NVT32-35 began to die by 6 d,and the peak of death appeared at 7-10 d.Fish challenged with rIHNV-ANV-EGFP began to die by 10 d.By 20 d,the mortalities of fish infected with rIHNV NVQ32-35,rIHNV NVT32-355 rIHNV-ANV-EGFP and wt IHNV HLJ-09 were 55%,60%,10%and 90%,respectively.Pathological sectioning results showed that the tissues(liver,heart)of the fish challenged with recombinant viruses exhibited pathological changes.However,the tissues(liver,brain,kidney and heart)of the fish challenged with wt IHNV HLJ-09 exhibited pathological changes.Additionally,the effect of recombinant viruses and wt IHNV HLJ-09 on the liver immune response were assessed.Our results indicated that amino acids 32EGDL35 within the NV played an important role in suppressing IFN1,Mx-1 and IL-6.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 viral copy numbers of rIHNV-NVQ32-35,rIHNV-NVT32-35,and rIHNV-△NV-EGFP were lower than wt IHNV HLJ-09,which testified when amino acids 32EGDL35 within the NV were mutated at the same time,the replication ability decreased with varying degrees,and the virulence of the virus decreased.