Studies On The Influence Of OsHV-1 Infection On The Iron Metabolism Of Scapharca Broughtonii

Iron ions are involved in various cellular metabolic processes as essential trace elements in almost all living forms.The life cycle of virus depends on the host cell,which also needs iron.It replication process is affected by the iron ion state in the host.The virus acquires iron ions by interfering with the iron metabolism of the host.In this study,Scapharca broughtonii was used as the research object.Foucsing on the S.broughtonii ferritin molecule,molecular biology,immunology and other related techniques were used to study the structure and biological function of S.broughtonii ferritin and discussed the pathogenic mechanism of the OsHV-1 interfering with the host’s iron metabolism.1.HKGs selection of S.broughtoniiIn this study,ten pairs of HKGs were selected,and the optimal HKGs were selected under different tissues,OsHV-1 challenge and temperature stress conditions.The results showed that the wildly used 18s and ACT HKGs were not stable under the experimental conditions.RL15 showed good stability under the above experimental conditions,and its M value was the lowest.It can be used as the first reference gene for Real-time quantitative PCR analysis.In addition,although the expression level of S18 and EF-1γ fluctuates greatly between tissues,it also has better stability under the above other experimental conditions.2.Ferritin protein structure and tissue distributionThrought the transcriptome data analysis,two types of ferritin genes in S.broughtonii tissue were preliminarily screened.A phylogenetic tree analysis was established and showed that two types of ferritin genes were clustered with cytosolic ferritin and mitochondrial ferritin,respectively,and named SbFn,Sb Mi Fn.There were alternative splice sites in the SbFn gene.It was found through cloning and sequencing analysis that the alternative splice sites were located in the intron regions 1 and 2 of the SbFn gene and were named SbSFn-1 and SbSFn-2,respectively.The conserved motif IRE exists in the 5’-UTR region of the SbFn gene.The three-dimensional structure of the SbFn gene is mainly consisted of four alpha helices and a typical Fe²⁺binding site,which is relatively conservative in evolution.SbFn was expressed in mantle,hepatopancreas,gill,haemolymph,foot and adductor muscle,with the highest expression in hepatopancreas,followed by gill and hemolymph.SbSFn-1 and SbSFn-2 possess three alpha helices and two alpha helices,respectively,and both have typical Fe²⁺ binding sites and typical iron oxidase sites.The expression level of SbSFn-1 was highest in the mantle,followed by hemolymph,hepatopancreas,foot,gill and adductor muscle.The SbSFn-2 gene has a higher expression level in the foot and haemolymph,while not in the mantle.The three-dimensional structure of Sb Mi Fn is more complex,mainly formed by six α-helices forming a cage structure,with a typical CXT binding site in the center of the cage structure,containing a oxidoreductase site.The Sb Mi Fn gene is not expressed in hepatopancreas and gill,and has the highest expression in the hemolymph.3.S.broughtonii ferritin functionIn this study,the Fe²⁺ binding activities of SbFn,Sb Mi Fn,SbSFn-1 and SbSFn-2 proteins were detected using phenazine,and the SbFn and Sb Mi Fn recombinant proteins showed significant Fe²⁺ binding activity,while the SbSFn-1 and SbSFn-2 recombinant proteins were not Fe²⁺ bound.Native-PAGE analysis revealed that SbFn and SbSFn-2 recombinant proteins exist many different types of multimers.The SbFn and SbSFn-2 recombinant proteins had different degrees of binding ability to Vibrio harveyi,Vibrio alginolyticus,Vibrio alginolyticus and Escherichia coli.External pathogens could acquire iron ions by intercepting S.broughtonii ferritin;SbFn,SbSFn-1 and SbSFn-2 could promote phagocytosis.SbFn and SbSFn-2 have a certain binding ability with burrow hemolymph,which may mediate the iron ion transport process in the tissue fluid through the presence of phagocytic cells.Surface ferritin or transferrin receptor mediates endocytosis of microorganisms.4.The pathogenicity of OsHV-1 infection to S.broughtoniiThe viral loads in the hepatopancreas,mantle,gill and haemolymph increased rapidly after S.broughtonii infection with OsHV-1,and the number of copies of OsHV-1 peaked in the tissue at 48 h,approximately 10⁵ copies/ng（total tissue DNA）.Afterwards,it remained at a relatively stable level.Meanwhile,different levels of lesions occurred in the S.broughtonii tissues.Transmission electron microscopic observations of the distribution of OsHV-1 in S.broughtonii after infection with OsHV-1 revealed a large number of virus particles in blood cells.Infected blood cell nuclear enlargement distortion,chromatin disintegration.The distribution of the OsHV-1 was also found in tissues such as mantle.5.Effect of OsHV-1 on tissue iron homeostasisAfter S.broughtonii infection with OsHV-1,SbFn gene expression levels in hepatopancreas and hemolymph of S.broughtonii were up-regulated,and began to decrease after 24 h.However,the expression levels were significantly lower than normal levels by 48 h;SbFn gene expression level was down-regulated in gill after 24 h infection significantly;SbFn gene expression levels in the mantle showed an upward trend at 48 h,after that the expression level began to decline.After S.broughtonii infection with OsHV-1,the content of iron in the tissue was changed.The total iron content in the hepatopancreas and hemolymph showed an upward trend after infection.The levels of hydrogen peroxide in the hepatopancreas,haemolymph,and gill showed an upward trend,which was basically consistent with the changes in tissue iron content.In summary,S.broughtonii ferritin plays an important role in the organization of iron stores and maintenance of iron homeostasis.OsHV-1 infection leads to changes in the expression of ferritin and affect the maintenance of iron homeostasis in S.broughtonii tissue.The increase of intracellular free iron content leads to an increase in reactive oxygen species in S.broughtonii tissue,resulting in tissue oxidative damage.The above results preliminarily clarified the pathogenic mechanism of OsHV-1 and laid an important theoretical basis for the prevention and control of OsHV-1.