The Effect Of VP4 And VP5 Proteins Of Chicken Infectious Bursal Disease Virus On The Natural Denfense Capability Of The Host In The Infection

Chicken infectious bursal disease(IBD)is an acute,highly contagious infectious disease,caused by infectious bursal disease virus(IBDV),which could cause the immunosuppression of chichen and brings a great harm to the poultry industry every year.VP4 and VP5 are two important non-structural proteins encoded by the genome of the virus which play important roles in the infection of the virus.1.The research on the immunosuppressive role of VP4 and VP5 proteins in IBD V infectionIn order to explore the immunosuppressive role of VP4 and VP5 proteins caused by IBDV infection,eukaryotic expression plasmids containing VP4 or VP5 gene were constructed in this study.Then,the plasmids were transfected into DF-1 cells,alone or stimulated by poly IC or infected with IBDV CV03 strains.The expression ofpattern recognition receptors and downstream transcription factors was detected by western blot.The expression of cytokines and antiviral factors was tested by fluorescence quantitative PCR.Experimental results showed that VP4 protein could promote the expression of TLR3 both in mRNA and protein levels,especially after the stimulation of poly IC or the infection of IBDV CV03.However,VP5 protein showed a significant inhibitory effect on the mRNA and protein expression of TLR3.The fold changes of TLR3 mRNA expression were higher compared with the low protein expression,and the protein expression of MDA5 was significantly inhibited by VP4 or VP5 protein after the infection of IBDV CV03.The further examination of downstream transcription factors showed that both VP4 and VP5 protein could promote the protein expression of IRF3.However,the effect was resversed after IBDV infection.The protein expression of IRF7 was significantly inhibited by VP5,and the mRNA expression of downstream effectors,such as type I IFN,IL-1 beta and IL-6,can also be suppressed by VP5 protein.In addition,the mRNA expression of Mx gene was visibly inhibited after infection with CV03 strain.The result showed that IBDV could achieve the immunosuppression through two pathways.One is block the downstream signal transmission through inhibiting the mRNA and protein expression of TLR3,one of the pattern recognition receptors,by VP5 protein,the other is repress the protein expression of MDA5.The detection of downstream transcriptional factor of pattern recognition receptors showed that the immunosuppressive effect caused by IBDV can be achieved by inhibiting the protein expression of IRF3,which was mainly executed by VP4 and VP5 proteins together.Simultaneously,IBDV can also achieve the immunosuppressive effect by inhibiting the expression of IRF7 both in mRNA and protein levels via VP5 protein.As a result,the immune suppression of IBDV in the interaction of IBDV and DF-1 cells mainly achieved by VP5 protein,and VP4 only played an auxiliary role.2.The effect of VP4 and VP5 proteins on non-inte rferon pathway in IBDV infectionTo explore the impact of VP4 and VP5 proteins on the host non-interferon pathway in IBDV infection,eukaryotic expression plasmids containing VP4 or VP5 gene were transfected into Vero cells,alone or infected with IBDV CV03 strains after transfection.Western-blot was carrid out to detect the expression of pattern recognition receptors(PRRs)and downstream transcription factors.The expression of cytokines and antivirus genes was detected by fluorescence quantitative RT-PCR.Experimental results showed that the expression of VP4 protein improves the protein expression of TLR3 and RIG-I to a certain extent after infection of IBDV CV03 strain.VP5 has a certain suppression in innate immune pathway mediated by TLR3 and RIG-I.The overexpression of VP5 in Vero cells not only significantly curbs the protein expression of IRF3 and its phosphorylation,but also restrains the expression of IRF7 and NF-KB obviously after IBDV infection.However,those effects were not observed after the overexpression of VP4.The inhibition of NF-?B by VP5 further leads to the suppression of TNF-a.The results illustrated that VP5 protein had a certain suppression in related signaling pathways(non-produce pathway of type I interferon),which was closely related to the inhibition of nuclear transcription factors and TNF-a.On the other hand,the influence of the overexpress VP4 and VP5 proteins in Vero cells on IBDV infection and replication is unclear,and it needed further exploration.3.The effect of VP4 and VP5 proteins on cell damage caused by viral infectionTo investigate the influence of VP4 and VP5 proteins on cell damage caused by IBDV infection,eukaryotic expression plasmids containing VP4 or VP5 gene were transfected into DF-1 cells,alone or stimulated by poly IC or infected with IBDV CV03 strains in this research.Then the level of reactive oxygen species(ROS)was detected by flow cytometry,and the enzymatic activity of SOD and NAG and the concentration of LD were also detected by kits.Experimenal results showed that VP4 and VP5 can stimulate the production of ROS in DF-1 cells,and increased the activity of SOD and the content of LD.When stimulated by poly IC or infected with IBDV CV03 strains,the level of ROS declined,but the activity of SOD still increased,although the increased amount reduced,and the content of LD increased,too.Neither VP4 nor VP5 had significant effects on the activity of NAG The results indicated that the cellular damage in the process of IBDV infection was likely caused by the excessive reaction of immune system of the host when it recognized the pathogen.The decrease of intracellular ROS and the weakened vitality of SOD,which was the antioxidant system,may ascribe to the reduced cell damage after IBDV infection indicating IBDV infection may restrain cell damage and death,which may be conducive to viral replication in DF-1 cells.