EEF1α Affects Replication Difference Between Very Virulent Infectious Bursal Disease Virus And Attenuated Strain

The infectious bursal disease（IBD）is a highly contact,acute,lethal infectious avian disease.The agent of disease is infectious bursal disease virus（IBDV）,which mainly infects immature B-lymphocytes of bursa of 3-6 weeks old poultry.IBDV serotypes can be divided into type I and type II,however,only serotype I causes disease in chicks.Two major mutations have occurred in the serotype I virus,resulting in three type strains:classic,variant,and very virulent strains.Very virulent IBDV（vvIBDV）can result in the death of chicks,while attenuated strain can not cause the death of chicks.Therefore,the difference in pathogenic mechanism between vvIBDV and attenuated strains deserves further study.B-lymphocyte of bursa is the ideal cell model to investigate the difference between vvIBDV and attenuated strains,since it can be infected by vvIBDV or attenuated strains in vitro.It has been reported that primary B-lymphocytes from chickens are susceptible to apoptosis in vitro.The apoptosis rate can reach more than 80%at 72 hours,but chicken CD40L can extend the survival of B lymphocytes for more than a few weeks.In this study,the eukaryotic expression vector of the CD40L functional domain was constructed and optimized by human codons for expression in 293T cells.The results showed that CD40L was successfully expressed and the purified CD40L was directly added to the medium of bursal B cells.The morphology of B cells,the cell activity,the apoptosis rate,and the quantity of cells were observed and detected at different time points.The results showed that the addition of CD40L can slightly increase the number of cells,enhance the activity of cells,and reduce the rate of apoptosis of B cells,but it cannot be cultured for a long time in vitro.In order to study and understand the mechanism of infectious bursal disease virus replication,this study carried out research on the interaction between IBDV and host.First,IBDV polyprotein was used for immunoprecipitation experiments,and the suspected bands were analyzed by mass spectrometry.It was found that one of the host proteins,eEF1α,interacts with the IBDV polyprotein.In order to further study the role of eEF1αin the replication of IBDV and the differential replication of vvIBDV and attenuated strains,we detected the changes of eEF1αin B cells infected with the same load of vvIBDV and attenuated strains at the level of transcription and protein expression.Both vvIBDV and attenuated strain infections promote the transcription of eEF1α,and the promotion of vvIBDV is higher than that of attenuated,but the expression level has not changed significantly.In order to investigate the effect of eEF1αon the replication of IBDV,effective siRNA target on chicken eEF1αwas screened.Since the down regulation of endogenous eEF1αin DF-1 cells,the expression of viral protein of attenuated Gt in DF-1 cells was reduced.And the TCID₅₀ titer of the virus can also be reduced.Besides,when knocking down of the endogenous eEF1αof the DT40 cells,both the amount of viral protein and the titer of ELD₅₀ of the vvIBDV can be obviously reduced.In contrast,overexpressing exogenous eEF1αin DT40 cells can slightly increase the amount of vvIBDV VP3 protein.It indicates that the replication of IBDV requires the participation of host protein eEF1α.Based on co-immunoprecipitation experiments,the results showed that eEF1αinteracts with vvIBDV VP3 protein,but there is no interaction with IBDV attenuated VP3.It means host protein eEF1αpossesses different properity in interaction with different IBDV VP3proteins.The confocal microscopy observed that eEF1αand vvIBDV VP3 could partially co-localize.Based on the sequence analysis,there are 4 amino acids differences between vvIBDV VP3 and attenuated VP3（28,226,235,250）.In order to further explore the determinant amino acids for the interaction between eEF1αand Gx-VP3,the VP3 protein was subjected to point mutations,and the co-immunoprecipitation experiments were performed.It was found that there was more than one determinant site of amino acids between eEF1αand Gx-VP3 interaction because none of the Gt-VP3 with any single mutation to corresponding of Gx-VP3amino acids could be co-immunoprecipitated by eEF1α.Moreover,the Gx-VP3 amount of co-immunoprecipitated by eEF1αwas significantly reduced with point mutation in 28,235,or 250 of Gx-VP3,demonstrating that these amino acids are important to interaction between eEF1αand IBDV VP3.In summary,the present study found that there was a difference in the interaction of eEF1αwith vvIBDV or attenuated IBDV,and that eEF1αcould affect the replication of IBDV.Because eEF1αis a translation elongation factor,it is speculated that eEF1αmay play a role in the translation of IBDV proteins.The underlying mechanism for these functions needs further investigation.