Mechanism Of Imbalance Of Ca²⁺ Homeostasis Mediated By Host Protein STIM1 In VvIBDV Pathogenesis

Infectious bursal disease?IBD?caused by infectious bursal disease virus?IBDV?is an acute and highly fatal immunosuppressive disease,which has a significant impact on the development of the world poultry industry.Bursa of Fabricius?BF?is the target organ of IBDV,and B lymphocytes are its target cells.However,study have shown that chickens after bursectomy could still survive,indicating that the absence of bursa did not lead to the death of the chicken.Clinical examination of IBDV-infected-chickens revealed that the infected chickens had typical"spotted kidney"symptoms,indicating that their kidneys were seriously damaged.There was also study showed that the serum uric acid concentration of vvIBDV-infected-chickens was significantly increased.As the kidney is an important organ in the metabolic process,and uric acid is the main end product of poultry metabolism,we speculated that infected chickens may die from severe metabolic disorders.Previous studies have found that the serum calcium(Ca²⁺)content of IBDV-infected-chickens was significantly lower than that of uninfected chickens.Ca²⁺homeostasis is extremely important for the normal operation of life activities.Therefore,this study was devoted to elucidating the pathogenic mechanism of vvIBDV from the perspective of ion metabolism.In our study,vvIBDV was used to infect 3-week old SPF chickens.We found that serum Ca²⁺levels of vvIBDV-infected-chickens were significantly lower than that of uninfected chickens.In vitro,the concentration of Ca²⁺in the endoplasmic reticulum?ER?of vvIBDV-infected DT40 cells was significantly increased.In the vvIBDV differentially expressed host protein screened by isobaric tags for relative and absolute quantification?iTRAQ?technology,the expression level of"Ca²⁺concentration receptor"in the ER-stromal molecule protein 1?STIM1?was significantly up-regulated.Western blot?WB?and immunofluorescence assay showed that vvIBDV infection significantly increased the protein expression of STIM1.Further studies found that down-regulated expression of STIM1 significantly reduced the Ca²⁺levels of the ER and significantly inhibited the replication of vvIBDV.Overexpression of STIM1significantly increased Ca²⁺levels in the ER and significantly promoted replication of vvIBDV.In order to explore the mechanism by which STIM1 regulates Ca²⁺levels of the ER and affects the replication of vvIBDV,co-immunoprecipitation?CO-IP?experiments showed that VP2 and VP4 of vvIBDV interacted with STIM1,activated STIM1,bound it to Orai1 protein on the cell membrane,opened calcium release-activated calcium?CRAC?channels,and allowed extracellular Ca²⁺to flow into the ER.In order to further confirm the role of CRAC channels in the process of vvIBDV infection,this study used specific inhibitor of CRAC channels for in vivo and in vitro tests.In vitro tests proved that the inhibition of CRAC channels significantly reduced the Ca²⁺levels in the ER and significantly inhibited the replication of vvIBDV.In vivo tests showed that inhibition of CRAC channels could stabilize the serum Ca²⁺levels of vvIBDV-infected-chickens,effectively improved metabolism,and thus reduced the mortality of chickens infected with vvIBDV.This study reveals the important pathogenic mechanism of vvIBDV,that is,host Ca²⁺homeostasis imbalance mediated by the host protein STIM1 caused by vvIBDV infection is an important factor for vvIBDV to kill chickens.Our study provides new insights for elucidating the pathogenesis of vvIBDV,and provides new ideas for the prevention and control of vvIBDV,and lay a research foundation for using CRAC channels as targets to treat host metabolic disorders.